Methods for treating inflammatory disorders using 2,4-pyrimidinediamine compounds

ABSTRACT

Methods for treating an inflammatory disorder and for inhibiting the production of IL-23 using 2,4-pyrimidinediamine compounds represented by formula (I):

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority benefit under 35 U.S.C. §119(e) to U.S.Provisional Patent Application No. 61/144,609, filed on Jan. 14, 2009,which is incorporated by reference in its entirety.

INTRODUCTION

Interleukin 23 (IL-23) is a heterodimeric cytokine consisting of twosubunits, p40 and p19. IL-23 plays a role in the inflammatory responseagainst infection, and has been implicated in the development ofmultiple sclerosis, inflammatory bowel disease, and cancer. Interleukin10 (IL-10) is an anti-inflammatory cytokine that is capable ofinhibiting the syntheses of various pro-inflammatory cytokines.

Inflammation is the complex biological response of vascular tissues toharmful stimuli, such as pathogens, damaged cells, or irritants.However, inflammation which runs unchecked can lead to a host ofdisorders, such as inflammatory arthritis, rheumatoid arthritis, hayfever, and atherosclerosis.

Inflammation can be classified as either acute or chronic. Acuteinflammation is the initial response of the body to harmful stimuli andis achieved by the increased movement of plasma and leukocytes from theblood into the injured tissues. A cascade of biochemical eventspropagates and matures the inflammatory response, involving the localvascular system, the immune system, and various cells within the injuredtissue. Acute inflammation is a short-term process which ischaracterized by the classic signs of inflammation—swelling, redness,pain, heat, and loss of function—due to the infiltration of the tissuesby plasma and leukocytes. It occurs as long as the injurious stimulus ispresent and ceases once the stimulus has been removed, broken down, orwalled off by scarring (fibrosis).

Prolonged inflammation, known as chronic inflammation, leads to aprogressive shift in the type of cells which are present at the site ofinflammation and is characterized by simultaneous destruction andhealing of the tissue from the inflammatory process. Chronicinflammation is a pathological condition characterized by concurrentactive inflammation, tissue destruction, and attempts at repair. Chronicinflammation is not characterized by the classic signs of acuteinflammation listed above. Instead, chronically inflamed tissue ischaracterized by the infiltration of mononuclear immune cells(monocytes, macrophages, lymphocytes, and plasma cells), tissuedestruction, and attempts at healing, which include angiogenesis andfibrosis. Endogenous causes include persistent acute inflammation.Exogenous causes are varied and include bacterial infection, prolongedexposure to chemical agents such as silica, or autoimmune reactions suchas rheumatoid arthritis.

Cells of the immune system use a signal cascade to mount an escalatingresponse to a real or perceived insult. The inflammatory responsebecomes pathogenic when the signal cascade is invoked inappropriately.For example, autoimmune diseases are the consequence of the immunesystem mounting a response against antigens which are intrinsic. Manyanti-inflammatory agents function by inhibiting the signal cascade, suchas by blocking intracellular or intercellular effectors.Glucocorticoids, for example, mimic the natural immune suppressant,cortisol, to block genes at the transcription level, and cyclo-oxygenaseinhibitors are small molecules that bind to and inhibit an enzyme thatprocesses an internal signal molecule in cells.

SUMMARY

Described herein are methods for treating inflammatory disorders using2,4-pyrimidinediamine compounds. Further described are methods forinhibiting the production of IL-23.

DETAILED DESCRIPTION

As used herein, the singular forms “a”, “an”, and “the” include pluralreferents unless the context clearly dictates otherwise. It is furthernoted that the claims may be drafted to exclude any optional element. Assuch, this statement is intended to serve as antecedent basis for use ofsuch exclusive terminology as “solely,” “only” and the like inconnection with the recitation of claim elements, or use of a “negative”limitation.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Described herein are 2,4-pyrimidinediamine compounds represented by thefollowing formula (I):

In the formula (I), X represents (CH₂)_(m), wherein m is an integer from1 to 5, and wherein one or more CH₂ are optionally replaced with O, S orN(R⁰) wherein R⁰ represents H, C₁-C₇ alkyl or C₃-C₈ cycloalkyl. In anembodiment, X represents (CH₂)₃ or (CH₂)₂. The term “alkyl,” as usedherein, denotes both straight- and branched-chain alkyls. Specificexamples of C₁-C₇ alkyls include, but are not limited to, methyl, ethyl,propyl, butyl, pentyl, hexyl and heptyl and their branched-chainisomers, e.g., isopropyl, isobutyl, sec-butyl, tert-butyl, etc. The term“cycloalkyl,” as used herein, denotes both monocyclic and bicyclicalkyls. Specific examples of C₃-C₈ cycloalkyl include, but are notlimited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl and cyclooctyl, their substituted isomers, e.g., methylcyclopentyl, methyl cyclohexyl, and their bicyclic isomers, e.g.,spiro[3,4]octyl and spiro[3,3]heptyl, and the like.

Y represents N(R¹), O or S, wherein R¹ represents H, C₁-C₇ alkyl orC₃-C₈ cycloalkyl.

In an embodiment, R¹ represents C₁-C₇ alkyl, such as CH₃. In a furtherembodiment, Y represents N(C₁-C₇ alkyl), such as N(CH₃).

In the formula (I), R¹¹, R¹², R¹³, and R¹⁴ each independently representsC₁-C₇ alkyl.

In the formula (I), the bond — may be attached to any ring atomcontained in X. In an embodiment, the bond — is attached to a carbonring atom.

One of skill in the art will appreciate that many of the compounds, aswell as the various compound species specifically described and/orillustrated herein, may exhibit tautomerism, conformational isomerism,geometric isomerism and/or optical isomerism. The term “tautomer” refersto alternate forms of a molecule that differ only in electronic bondingof atoms and/or the position of a proton, such as enol-keto andimine-enamine tautomers, or the tautomeric forms of heteroaryl groupscontaining a —N═C(H)—NH— ring atom arrangement, such as pyrazoles,imidazoles, benzimidazoles, triazoles, and tetrazoles. A person ofordinary skill in the art would recognize that other tautomeric ringatom arrangements are possible. For example, the disclosed compounds mayinclude a stereogenic element, such as one or more chiral centers and/ordouble bonds and, as a consequence, may exist as stereoisomers, such ascis-trans isomers, E and Z isomers, enantiomers and diastereomers andmixtures thereof, including racemic and optically active mixtures. Theterm “stereoisomer” refers to isomeric molecules whose atomicconnectivity is the same but whose atomic arrangement in space isdifferent. The term “enantiomers” refers to compound that arestereoisomers that are nonsuperimposable minor images of each other. Theterm “diastereomers” refers to a steroisomers not related as mirrorimages. The term “racemic” or “racemate” refers to a mixture ofequimolar quantities of two enantiomeric species.

In certain embodiments, the disclosed 2,4-pyrimidinediamine compoundsare in the form of pharmaceutically acceptable salts. Generally,pharmaceutically acceptable salts are those that retain substantiallyone or more of the desired pharmacological activities of the parentcompound and which are suitable for administration to a subject.Examples of the presently disclosed compounds include at least one basicamino group. Thus, pharmaceutically acceptable salts of such compoundsinclude acid addition salts formed with inorganic acids or organicacids. Inorganic acids suitable for forming pharmaceutically acceptableacid addition salts with the present compounds include, by way ofexample, hydrohalide acids (hydrochloric acid, hydrobromic acid,hydroiodic acid), sulfuric acid, nitric acid, phosphoric acid, and thelike. Organic acids suitable for forming pharmaceutically acceptableacid addition salts include, without limitation, acetic acid,trifluoroacetic acid, propionic acid, hexanoic acid,cyclopentanepropionic acid, glycolic acid, oxalic acid, pyruvic acid,lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, palmitic acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,alkylsulfonic acids (such as, methanesulfonic acid, ethanesulfonic acid,1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid), arylsulfonicacids (such as benzenesulfonic acid, 4 chlorobenzenesulfonic acid,2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonicacid), 3-phenylpropionic acid, trimethylacetic acid, tertiarybutylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid,hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, andthe like.

In the formula (I), — when attached to a stereogenic element, such as achiral center or double bond, indicates that the bond can be attached ineither configuration. Further, — represents a bond of either possiblerelative or absolute stereochemistry. In compounds having a chiralcenter, the symbol — is used to indicate that the chiral center may havethe configuration, R or S, or both.

In an embodiment, the ring having X and Y has the following formula:

In a further embodiment, Y in the above formulae is N(R¹), such as,N(CH₃).

In the formula (I), p is 0 or 1.

In the formula (I), R² represents H, C₁-C₇ alkyl or C₂-C₈ alkanoyl. R⁴represents H, C₁-C₇ alkyl or C₂-C₈ alkanoyl. In an embodiment, R² and R⁴are H.

In the formula (I), R³ represents H, halogen, cyano, nitro, (C₁-C₇alkoxy)carbonyl, C₁-C₇ alkyl, C₁-C₇ haloalkyl, C₁-C₇ alkoxy or C₁-C₇haloalkoxy. The term “alkoxy,” as used herein, denotes groups havingstraight- or branched-chain alkyls. Specific examples of C₁-C₇ alkoxyinclude methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy andheptyloxy and their branched-chain isomers, e.g., isopropoxy, isobutoxy,sec-butyloxy, tert-butyloxy, etc. The term “halo-,” as used herein,denotes halogens including fluoro-, chloro-, bromo- and iodo-, and forexample, fluoro- and chloro-. The haloalkyl and haloalkoxy may have oneor more halogen substituent(s) at any suitable position(s) on the alkylchain.

Specific examples of suitable haloalkyl include, but are not limited to,chloromethyl, dichloromethyl, trichloromethyl, bromomethyl,fluoromethyl, difluoromethyl, trifluoromethyl, 1-fluoroethyl,1-chloroethyl, 2-chloroethyl, 2,2,2-trichloroethyl,2,2,2-trifluoroethyl, 1,2-dichloroethyl, 1-chloropropyl, 3-chloropropyl,1-chlorobutyl, 1-chloropentyl, 1-chlorohexyl, 4-chlorohexyl,4-chlorobutyl and the like.

Specific examples of suitable haloalkoxy include, but are not limitedto, chloromethoxy, dichloromethoxy, trichloromethoxy, bromomethoxy,fluoromethoxy, difluoromethoxy, trifluoromethoxy, 1-fluoroethoxy,1-chloroethoxy, 2-chloroethoxy, 2,2,2-trichloroethoxy,2,2,2-trifluoroethoxy, 1,2-dichloroethoxy, 1-chloropropoxy,3-chloropropoxy, 1-chlorobutoxy, 1-chloropentyloxy, 1-chlorohexyloxy,4-chlorohexyloxy, 4-chlorobutoxy and the like.

In an embodiment, C₁-C₇ haloalkyl is CHF₂ or CF₃. In a furtherembodiment, R³ represents F, CF₃, CN, NO₂, or COOCH₂CH₃. In anotherembodiment, R³ represents F.

In the formula (I), R⁵ represents substituted aryl or heteroaryl. Theterm “substituted” or “substituent,” as used herein, denotes an atom orgroup of bonded atoms replaced a hydrogen atom in a parent molecule. Inan embodiment, aryl or heteroaryl encompassed by R⁵ has 6 to 14 ringatoms. The term “aryl,” as used herein, denotes monocyclic andpolycyclic aromatic systems. In an embodiment, the aryl encompassed byR⁵ represents substituted phenyl or naphthyl. The term “heteroaryl,” asused herein, denotes monocyclic and polycyclic aromatic systems havingone or more, and for example, up to 5, heteroatoms selected from thegroup consisting of O, S and N. In an embodiment, the heteroarylcontains one or two O, S or N(R⁰¹) wherein R⁰¹ represents H, C₁-C₇ alkylor C₃-C₈ cycloalkyl. Specific examples of suitable heteroaryls include,but are not limited to, thienyl, furyl, furazanyl, pyrrolyl, imidazolyl,pyrazolyl, isothiazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl,pyridazinyl, benzofuranyl, isobenzofuranyl, indolizinyl, indolyl,isoindolyl, 1H-indazolyl, purinyl, quinolyl, isoquinolyl, phthalazinyl,naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, andthe like.

When R⁵ represents substituted heteroaryl, the heteroaryl may beconnected to the N2 atom through any atom in the ring therein, and forexample, through a carbon atom.

In an embodiment, the substituted aryl or heteroaryl encompassed by R⁵are disubstituted. In a further embodiment, the disubstitution occurs atthe positions which are meta- and para- to the position through whichthe aryl or heteroaryl is connected to the N atom. In anotherembodiment, R⁵ represents substituted aryl or heteroaryl having 6 ringatoms.

In a further embodiment, R⁵ represents the following formula:

wherein R⁶ and R⁷ each independently represents H, halogen, cyano, C₁-C₇alkyl, C₁-C₇ haloalkyl, C₁-C₇ alkoxy, C₁-C₇ haloalkoxy, C₃-C₈cycloalkyl, —SO₂—C₃-C₈ cycloalkyl, C₃-C₈ cycloalkyl-SO₂—R⁸, C₃-C₈cycloalkyl, —SO₂—C₃-C₈ cycloalkenyl, C₃-C₈ cycloalkenyl-SO₂—R⁸, aryl,—SO₂-aryl, aryl-SO₂—R⁸, a heterocyclic group, a —SO₂-heterocyclic group,a heterocyclic-SO₂—R⁸ group, or —SO₂—NR⁹R¹⁰; or R⁶ and R⁷ are combinedto form a ring together with the carbon atoms to which they are bonded.R⁸ and R⁹ each independently represents C₁-C₇ alkyl or C₃-C₈ cycloalkyl,and R¹⁰ represents H, C₁-C₇ alkyl or C₃-C₈ cycloalkyl.

In a further embodiment, R⁵ represents the following formula:

wherein R⁶ represents halogen, cyano, C₁-C₇ alkyl, C₁-C₇ haloalkyl,C₁-C₇ alkoxy or C₁-C₇ haloalkoxy; and R⁷ represents H, C₁-C₇ alkyl,C₁-C₇ haloalkyl, C₁-C₇ alkoxy, C₁-C₇ haloalkoxy, C₃-C₈ cycloalkyl,—SO₂—C₃-C₈ cycloalkyl, C₃-C₈ cycloalkyl-SO₂—R⁸, C₃-C₈ cycloalkyl,—SO₂—C₃-C₈ cycloalkenyl, C₃-C₈ cycloalkenyl-SO₂—R⁸, aryl, —SO₂-aryl,aryl-SO₂—R⁸, a heterocyclic group, a —SO₂-heterocyclic group, aheterocyclic-SO₂—R⁸ group, or —SO₂—NR⁹R¹⁰. R⁸ and R⁹ each independentlyrepresents C₁-C₇ alkyl or C₃-C₈ cycloalkyl, and R¹⁰ represents H, C₁-C₇alkyl or C₃-C₈ cycloalkyl.

In an embodiment, R⁶ represents Cl, CN, CF₃, or OCHF₂.

The term “cycloalkenyl,” as used herein, denotes both monocyclic andbicyclic alkenyls. Specific examples of C₃-C₈ cycloalkenyls include, butare not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclopentadienyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl,cycloheptadienyl, cycloheptatrienyl, cyclooctenyl, cyclooctadienyl,cyclooctatrienyl and their substituted derivatives, e.g., methylcyclobutenyl, methyl cyclopentenyl, methyl cyclohexenyl, and the like,and their bicyclic isomers.

The term “heterocyclic group,” as used herein, denotes aromatic,aliphatic and unsaturated, mono- and poly-cyclic groups having one ormore heteroatoms. The polycyclic group may contain two or more spiro,fused or bridged rings.

In an embodiment, the heterocyclic group has 1 to 3 rings. In anotherembodiment, the heterocyclic group has 3 to 14 carbon ring atoms, atleast one of which is replaced with a heteroatom, such as O, S or N(R⁰¹)wherein R⁰¹ represents H, C₁-C₇ alkyl or C₃-C₈ cycloalkyl.

The heterocyclic group may be attached through any ring atom therein. Inan embodiment, R⁷ represents a heterocyclic group which is attachedthrough a N ring atom.

Specific examples of suitable heterocyclic groups include, but are notlimited to, aziridinyl, oxiranyl, thiiranyl, azetidinyl, oxetanyl,thietanyl, pyrrolidinyl, tetrahydrofuryl, tetrahydrothienyl, piperidyl,tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydropyridyl,tetrahydroquinolyl, tetrahydroisoquinolyl, morpholinyl, thiomorpholinyl,piperazinyl, dioxolanyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl,thiazolidinyl, pyrazolidinyl, 2H-pyrrolyl, pyrrolinyl, imidazolinyl,pyrazolinyl, dihydrofuryl, dihydrothienyl, dihydropyranyl,dihydrothiopyranyl, dihydropyridyl, dihydroquinolyl, dihydroisoquinolyl,indolinyl, isoindolinyl, furyl, furazanyl, pyranyl, thienyl, pyrrolyl,pyrazolyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, oxadiazolyl,isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyridazinyl, pyrimidinyl,pyrazinyl, triazinyl, benzopyranyl, benzofuranyl, indolyl, quinolinyland the like. In these groups, the attachment may occur at a hetero ringatom or a carbon ring atom therein.

In an embodiment, R⁷ has the following formula:

wherein q represents 0 or 1, and R¹¹ represents H or C₁-C₇ alkyl. In afurther embodiment, R⁸ represents methyl.

In another embodiment, R⁷ represents one of the following formulae:

Alternatively, R⁶ and R⁷ may be combined to form a heterocyclic ringtogether with the carbon atoms to which they are bonded. For example, R⁶and R⁷ may be combined to form one of the following rings together withthe carbon atoms to which they are bonded:

R¹¹⁰ is H, C₁-C₇ alkyl or C₂-C₈ alkanoyl. R¹¹¹ is H, C₁-C₇ alkyl orC₂-C₈ alkanoyl. In certain embodiments, R¹¹⁰ is H or C₁-C₇ alkyl. Incertain embodiments, R¹¹¹ is H or C₁-C₇ alkyl.

Alternatively, R⁶ and R⁷ may be combined to form a heterocyclic ringtogether with the carbon atoms to which they are bonded. For example, R⁶and R⁷ may be combined to form the following ring together with thecarbon atoms to which they are bonded:

In one embodiment, the 2,4-pyrimidinediamine compound described hereinhas one of the following formulae (II) to (V):

R¹, R³, R⁵, R⁶, R⁷ and p in the above formulae (II) to (V) have the samemeanings as defined with respect to formula (I). In one embodiment, R⁶represents halogen or C₁-C₇ haloalkyl, and for example, Cl or CF₃.

In another embodiment, the 2,4-pyrimidinediamine compound describedherein has the following formula (VI) or (VII):

wherein:

R¹ has the same meaning as defined in the formula (I);

R³ represents F, CF₃, CN, NO₂ or COOCH₂CH₃;

R⁶ represents CF₃, Cl, CN or OCHF₂; and/or

R⁷ represents the following formula:

wherein R¹¹ represents H or C₁-C₇ alkyl; and q is 0 or 1,

wherein:

R¹ has the same meaning as defined in the formula (I);

R³ represents F, CF₃, CN, NO₂ or COOCH₂CH₃;

R⁶ represents CF₃, Cl, CN or OCHF₂; and/or

R⁷ represents the following formula:

wherein R¹¹ represents H or C₁-C₇ alkyl; and q is 0 or 1.

In one embodiment, the compound has the formula (I), wherein Xrepresents (CH₂)₂ or (CH₂)₃, Y represents N(R¹) wherein R¹ represents H,C₁-C₇ alkyl or C₃-C₈ cycloalkyl, R¹¹, R¹², R¹³, and R¹⁴ eachindependently represents C₁-C₇ alkyl, p is 0 or 1, R² and R⁴ are H, R³represents halogen, cyano, nitro, (C₁-C₇ alkoxy)carbonyl, or C₁-C₇haloalkyl, R⁵ represents the following formula:

R⁶ represents halogen, cyano, C₁-C₇ haloalkyl, or C₁-C₇ haloalkoxy, andR⁷ represents H, C₁-C₇ alkoxy, a heterocyclic group, a —SO₂-heterocyclicgroup, or a heterocyclic-SO₂—R⁸ group, wherein R⁸ represents C₁-C₇ alkylor C₃-C₈ cycloalkyl. In a further embodiment, R³ represents F. Inanother further embodiment, R⁶ represents Cl, CN, CF₃, or OCHF₂. Inanother further embodiment, R⁷ represents:

H, C₁-C₇ alkoxy,

wherein q represents 0 or 1, and R¹¹ represents H or C₁-C₇ alkyl.

In another embodiment, the compound has the formula (I), wherein Xrepresents (CH₂)₂ or (CH₂)₃, Y represents N(R¹) wherein R¹ represents H,C₁-C₇ alkyl or C₃-C₈ cycloalkyl, R¹¹, R¹², R¹³, and R¹⁴ eachindependently represents C₁-C₇ alkyl, p is 0 or 1, R² and R⁴ are H, R³represents halogen, cyano, nitro, (C₁-C₇ alkoxy)carbonyl, or C₁-C₇haloalkyl, R⁵ represents the following formula:

R⁶ and R⁷ are combined to form one of the following rings together withthe carbon atoms to which they are bonded:

In a further embodiment, R³ represents F.

In another embodiment, the compound has the formula (I) having an endoconfiguration, wherein X represents (CH₂)₂ or (CH₂)₃, Y represents N(R¹)wherein R¹ represents H, C₁-C₇ alkyl or C₃-C₈ cycloalkyl, R¹¹, R¹², R¹³,and R¹⁴ each independently represents C₁-C₇ alkyl, p is 0, R² and R⁴ areH, R³ represents F, CF₃, CN, NO₂, or COOCH₂CH₃, R⁵ represents theformula

R⁶ represents Cl, CN, CF₃, or OCHF₂, and R⁷ represents:

H, OCH₃,

wherein q represents 0 or 1, and R¹¹ represents H or C₁-C₇ alkyl. In afurther embodiment, R³ represents F.

In another embodiment, the compound has the formula (I) having an endoconfiguration, wherein X represents (CH₂)₂ or (CH₂)₃, Y represents N(R¹)wherein R¹ represents H, C₁-C₇ alkyl or C₃-C₈ cycloalkyl, R¹¹, R¹², R¹³,and R¹⁴ each independently represents C₁-C₇ alkyl, p is 0, R² and R⁴ areH, R³ represents F, CF₃, CN, NO₂, or COOCH₂CH₃, R⁵ represents theformula

R⁶ and R⁷ are combined to form one of the following rings together withthe carbon atoms to which they are bonded:

In a further embodiment, R³ represents F. In another further embodiment,R⁶ and R⁷ are combined to form, together with the carbon atoms to whichthey are bonded,

The disclosed compounds are further described in the followingnon-limiting embodiments.

Embodiment 1

A compound according to the following formula (I):

whereinX represents (CH₂)_(m) wherein m is an integer from 1 to 4, and whereinone or more CH₂ is optionally replaced with O, S or N(R⁰, wherein R⁰represents H, C₁-C₇ alkyl or C₃-C₈ cycloalkyl;Y represents N(R¹), O or S, wherein R¹ represents H, C₁-C₇ alkyl orC₃-C₈ cycloalkyl;R¹¹, R¹², R¹³, and R¹⁴ each independently represents C₁-C₇ alkyl;p is 0 or 1;R² represents H, C₁-C₇ alkyl or C₂-C₈ alkanoyl;R³ represents H, halogen, cyano, nitro, (C₁-C₇ alkoxy)carbonyl, C₁-C₇alkyl, C₁-C₇ haloalkyl, C₁-C₇ alkoxy or C₁-C₇ haloalkoxy;R⁴ represents H, C₁-C₇ alkyl or C₂-C₈ alkanoyl; andR⁵ represents substituted aryl or heteroaryl.

Embodiment 2

A compound according to Embodiment 1, wherein X represents (CH₂)₂ or(CH₂)₃.

Embodiment 3

A compound according to Embodiment 1 or 2, wherein Y represents N(R¹)and wherein R¹ represents H, C₁-C₇ alkyl.

Embodiment 4

A compound according to Embodiment 3, wherein R¹ represents methyl.

Embodiment 5

A compound according to any one of the proceeding Embodiments, R¹¹, R¹²,R¹³, and R¹⁴ each independently represents methyl.

Embodiment 6

A compound according to any one of the proceeding Embodiments, wherein pis 0 or 1.

Embodiment 7

A compound according to any one of the proceeding Embodiments, wherein pis 0.

Embodiment 8

A compound according to any one of the proceeding Embodiments, whereinR² and R⁴ each represents H.

Embodiment 9

A compound according to any one of the proceeding Embodiments, whereinR³ represents H, halogen, cyano, nitro, (C₁-C₇ alkoxy)carbonyl, C₁-C₇alkyl, C₁-C₇ haloalkyl, C₁-C₇ alkoxy or C₁-C₇ haloalkoxy.

Embodiment 10

A compound according to any one of the proceeding Embodiments, whereinR³ represents F, CF₃, CN, NO₂, or COOCH₂CH₃.

Embodiment 11

A compound according to any one of the proceeding Embodiments, whereinR³ represents F.

Embodiment 12

A compound according to any one of the proceeding Embodiments, whereinR⁵ represents substituted aryl or heteroaryl having 6 ring atoms.

Embodiment 13

A compound according to any one of the proceeding Embodiments, whereinR⁵ represents one of the following formulae:

wherein R⁶ represents halogen, cyano, C₁-C₇ alkyl, C₁-C₇ haloalkyl,C₁-C₇ alkoxy or C₁-C₇ haloalkoxy; andR⁷ represents H, C₁-C₇ alkyl, C₁-C₇ haloalkyl, C₁-C₇ alkoxy, C₁-C₇haloalkoxy, C₃-C₈ cycloalkyl, —SO₂—C₃-C₈ cycloalkyl, C₃-C₈cycloalkyl-SO₂—R⁸, C₃-C₈ cycloalkyl, —SO₂—C₃-C₈ cycloalkenyl, C₃-C₈cycloalkenyl-SO₂—R⁸, aryl, —SO₂-aryl, aryl-SO₂—R⁸, a heterocyclic group,a —SO₂-heterocyclic group, a heterocyclic-SO₂—R⁸ group, or —SO₂—NR⁹R¹⁰,wherein R⁸ and R⁹ each independently represents C₁-C₇ alkyl or C₃-C₈cycloalkyl, and R¹⁰ represents H, C₁-C₇ alkyl or C₃-C₈ cycloalkyl; orR⁶ and R⁷ are combined to form a ring together with the carbon atoms towhich they are bonded.

Embodiment 14

A compound according to Embodiment 13, wherein the heterocyclic groupencompassed by R⁷ is aromatic, aliphatic or unsaturated.

Embodiment 15

A compound according to Embodiment 13, wherein the heterocyclic groupencompassed by R⁷ is spiro, fused or bridged.

Embodiment 16

A compound according to Embodiment 13, wherein the heterocyclic groupencompassed by R⁷ has 3 to 14 carbon ring atoms, at least one of whichis replaced with O, S or N(R⁰¹) wherein R⁰¹ represents H, C₁-C₇ alkyl orC₃-C₈ cycloalkyl.

Embodiment 17

A compound according to Embodiment 13, wherein the heterocyclic groupencompassed by R⁷ has 1 to 3 rings.

Embodiment 18

A compound according to Embodiment 13, wherein the heterocyclic groupencompassed by R⁷ is selected from the group consisting of aziridinyl,oxiranyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl,tetrahydrofuryl, tetrahydrothienyl, piperidyl, tetrahydropyranyl,tetrahydrothiopyranyl, tetrahydropyridyl, tetrahydroquinolyl,tetrahydroisoquinolyl, morpholinyl, thiomorpholinyl, piperazinyl,dioxolanyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl,pyrazolidinyl, 2H-pyrrolyl, pyrrolinyl, imidazolinyl, pyrazolinyl,dihydrofuryl, dihydrothienyl, dihydropyranyl, dihydrothiopyranyl,dihydropyridyl, dihydroquinolyl, dihydroisoquinolyl, indolinyl,isoindolinyl, furyl, furazanyl, pyranyl, thienyl, pyrrolyl, pyrazolyl,imidazolyl, triazolyl, tetrazolyl, oxazolyl, oxadiazolyl, isoxazolyl,thiazolyl, isothiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl,triazinyl, benzopyranyl, benzofuranyl, indolyl, and quinolinyl.

Embodiment 19

A compound according to any one of the proceeding Embodiments 13 to 18,wherein R⁶ represents Cl, CN, CF₃, or OCHF₂.

Embodiment 20

A compound according to Embodiment 19, wherein R⁶ represents C₁ or CF₃.

Embodiment 21

A compound according to Embodiment 20, wherein R⁶ represents CF₃.

Embodiment 22

A compound according to any one of the proceeding Embodiments 13 to 21,wherein R⁷ represents the following formula:

H, C₁-C₇ alkoxy,

wherein q represents 0 or 1, and R¹¹ represents H or C₁-C₇ alkyl.

Embodiment 23

A compound according to Embodiment 22, wherein R⁷ represents thefollowing formula:

Embodiment 24

A compound according to Embodiment 23, wherein R⁸ represents methyl orcyclopropyl.

Embodiment 25

A compound according to Embodiment 13, wherein R⁶ and R⁷ are combined toform one of the following rings together with the carbon atoms to whichthey are bonded:

Embodiment 26

A compound according to Embodiment 25, wherein R⁶ and R⁷ are combined toform, together with the carbon atoms to which they are bonded,

Non-limiting specific examples of the compounds described herein are asfollows:

The present embodiments also provide a compound of formula (VIII):

wherein

R¹ represents H, C₁-C₇ alkyl or C₃-C₈ cycloalkyl;

R¹¹, R¹², R¹³, and R¹⁴ each independently represents C₁-C₇ alkyl;

z is 0 or 1;

R² represents H, C₁-C₇ alkyl or C₂-C₈ alkanoyl;

R³ represents H, halogen, cyano, nitro, (C₁-C₇ alkoxy)carbonyl, C₁-C₇alkyl, C₁-C₇ haloalkyl, C₁-C₇ alkoxy or C₁-C₇ haloalkoxy;

R⁴ represents H, C₁-C₇ alkyl or C₂-C₈ alkanoyl;

p is 0 or 1;

Q¹, Q², Q³, Q⁴, and Q⁵ are independently selected from C and N;

R⁶, R⁷, R¹⁰⁰, R¹⁰¹, and R¹⁰² independently selected from H, halogen,cyano, C₁-C₇ alkyl, C₁-C₇ haloalkyl, C₁-C₇ alkoxy, C₁-C₇ haloalkoxy,C₃-C₈ cycloalkyl, —SO₂—C₃-C₈ cycloalkyl, C₃-C₈ cycloalkyl-SO₂—R⁸, C₃-C₈cycloalkyl, —SO₂—C₃-C₈ cycloalkenyl, C₃-C₈ cycloalkenyl-SO₂—R⁸, aryl,—SO₂-aryl, aryl-SO₂—R⁸, a heterocyclic group, a —SO₂-heterocyclic group,a heterocyclic-SO₂—R⁸ group, or —SO₂—NR⁹R¹⁰; or

any two of R⁶, and R⁷, R¹⁰⁰, R¹⁰¹, R¹⁰² that are vicinal are combined toform a ring together with the carbon atoms to which they are bonded; and

any of R⁶, R⁷, R¹⁰⁰, R¹⁰¹, and R¹⁰² is absent to satisfy valencerequirements;

wherein R⁸ and R⁹ each independently represents C₁-C₇ alkyl or C₃-C₈cycloalkyl, and

R¹⁰ represents H, C₁-C₇ alkyl or C₃-C₈ cycloalkyl.

The present embodiments also provide a compound of formula (IX):

wherein

R¹ represents H, C₁-C₇ alkyl or C₃-C₈ cycloalkyl;

R¹¹, R¹², R¹³, and R¹⁴ each independently represents C₁-C₇ alkyl;

z is 0 or 1;

R² represents H, C₁-C₇ alkyl or C₂-C₈ alkanoyl;

R³ represents H, halogen, cyano, nitro, (C₁-C₇ alkoxy)carbonyl, C₁-C₇alkyl, C₁-C₇ haloalkyl, C₁-C₇ alkoxy or C₁-C₇ haloalkoxy;

R⁴ represents H, C₁-C₇ alkyl or C₂-C₈ alkanoyl;

Q¹, Q², Q³, Q⁴, and Q⁵ are independently selected from C and N;

R⁶, R⁷, R¹⁰⁰, R¹⁰¹, and R¹⁰² independently selected from H, halogen,cyano, C₁-C₇ alkyl, C₁-C₇ haloalkyl, C₁-C₇ alkoxy, C₁-C₇ haloalkoxy,C₃-C₈ cycloalkyl, —SO₂—C₃-C₈ cycloalkyl, C₃-C₈ cycloalkyl-SO₂—R⁸, C₃-C₈cycloalkyl, —SO₂—C₃-C₈ cycloalkenyl, C₃-C₈ cycloalkenyl-SO₂—R⁸, aryl,—SO₂-aryl, aryl-SO₂—R⁸, a heterocyclic group, a —SO₂-heterocyclic group,a heterocyclic-SO₂—R⁸ group, or —SO₂—NR⁹R¹⁰; or

any two of R⁶, R⁷, R¹⁰⁰, R¹⁰¹, and R¹⁰² that are vicinal are combined toform a ring together with the carbon atoms to which they are bonded; and

any of R⁶, R⁷, R¹⁰⁰, R¹⁰¹, and R¹⁰² is absent to satisfy valencerequirements;

wherein R⁸ and R⁹ each independently represents C₁-C₇ alkyl or C₃-C₈cycloalkyl, and R¹⁰ represents H, C₁-C₇ alkyl or C₃-C₈ cycloalkyl.

The present embodiments also provide a compound of formula (X):

wherein

R¹ represents H, C₁-C₇ alkyl or C₃-C₈ cycloalkyl;

R¹¹, R¹², R¹³, and R¹⁴ each independently represents C₁-C₇ alkyl;

R² represents H, C₁-C₇ alkyl or C₂-C₈ alkanoyl;

R³ represents H, halogen, cyano, nitro, (C₁-C₇ alkoxy)carbonyl, C₁-C₇alkyl, C₁-C₇ haloalkyl, C₁-C₇ alkoxy or C₁-C₇ haloalkoxy;

R⁴ represents H, C₁-C₇ alkyl or C₂-C₈ alkanoyl;

Q¹, Q², Q³, Q⁴, and Q⁵ are independently selected from C and N;

R⁶, R⁷, R¹⁰⁰, R¹⁰¹, and R¹⁰² are independently selected from H, halogen,cyano, C₁-C₇ alkyl, C₁-C₇ haloalkyl, C₁-C₇ alkoxy, C₁-C₇ haloalkoxy,C₃-C₈ cycloalkyl, —SO₂—C₃-C₈ cycloalkyl, C₃-C₈ cycloalkyl-SO₂—R⁸, C₃-C₈cycloalkyl, —SO₂—C₃-C₈ cycloalkenyl, C₃-C₈ cycloalkenyl-SO₂—R⁸, aryl,—SO₂-aryl, aryl-SO₂—R⁸, a heterocyclic group, a —SO₂-heterocyclic group,a heterocyclic-SO₂—R⁸ group, or —SO₂—NR⁹R¹⁰; or

any two of R⁶ and R⁷, R¹⁰⁰, R¹⁰¹, and R¹⁰² that are vicinal are combinedto form a ring together with the carbon atoms to which they are bonded;and

any of R⁶, R⁷, R¹⁰⁰, R¹⁰¹, and R¹⁰² is absent to satisfy valencerequirements;

wherein R⁸ and R⁹ each independently represents C₁-C₇ alkyl or C₃-C₈cycloalkyl, and

R¹⁰ represents H, C₁-C₇ alkyl or C₃-C₈ cycloalkyl.

The present embodiments also provide a compound of formula (XI):

wherein

R¹ represents H, C₁-C₇ alkyl or C₃-C₈ cycloalkyl;

R¹¹, R¹², R¹³, and R¹⁴ each independently represents C₁-C₇ alkyl;

R² represents H, C₁-C₇ alkyl or C₂-C₈ alkanoyl;

R³ represents H, halogen, cyano, nitro, (C₁-C₇ alkoxy)carbonyl, C₁-C₇alkyl, C₁-C₇ haloalkyl, C₁-C₇ alkoxy or C₁-C₇ haloalkoxy;

R⁴ represents H, C₁-C₇ alkyl or C₂-C₈ alkanoyl;

Q¹, Q², Q³, Q⁴, and Q⁵ are independently selected from C and N;

R⁶, R⁷, R¹⁰⁰, R¹⁰¹, and R¹⁰² are independently selected from H, halogen,cyano, C₁-C₇ alkyl, C₁-C₇ haloalkyl, C₁-C₇ alkoxy, C₁-C₇ haloalkoxy,C₃-C₈ cycloalkyl, —SO₂—C₃-C₈ cycloalkyl, C₃-C₈ cycloalkyl-SO₂—R⁸, C₃-C₈cycloalkyl, —SO₂—C₃-C₈ cycloalkenyl, C₃-C₈ cycloalkenyl-SO₂—R⁸, aryl,—SO₂-aryl, aryl-SO₂—R⁸, a heterocyclic group, a —SO₂-heterocyclic group,a heterocyclic-SO₂—R⁸ group, or —SO₂—NR⁹R¹⁰; or

any two of R⁶, R⁷, R¹⁰⁰, R¹⁰¹, and R¹⁰² that are vicinal are combined toform a ring together with the carbon atoms to which they are bonded; and

any of R⁶, R⁷, R¹⁰⁰, R¹⁰¹, and R¹⁰² is absent to satisfy valencerequirements;

wherein R⁸ and R⁹ each independently represents C₁-C₇ alkyl or C₃-C₈cycloalkyl, and

R¹⁰ represents H, C₁-C₇ alkyl or C₃-C₈ cycloalkyl.

In a certain embodiment, the 2,4-pyrimidinediamine compounds describedherein can be synthesized from substituted or unsubstituted uracils asillustrated in Scheme I, below.

In Scheme I, X, Y, p, R¹¹, R¹², R¹³, R¹⁴, R², R³, R⁴ and R⁵ are the sameas defined with respect to the formula (I). According to Scheme I,uracil A-1 is dihalogenated at the 2- and 4-positions using a standarddehydrating-halogenating agent such as POCl₃ under standard conditionsto yield 2,4-dichloropyrimidine A-2. Depending upon the nature of the R³substituent in pyrimidinediamine A-2, the chloride at the C4 position ismore reactive towards nucleophiles than the chloride at the C2 position.This differential reactivity can be exploited by first reacting2,4-dichloropyrimidine A-2 with one equivalent of amine A-3, yielding4N-substituted-2-chloro-4-pyrimidineamine A-4, followed by amine A-5 toyield a 2,4-pyrimidinediamine derivative A-6.

Typically, the C4 halide is more reactive towards nucleophiles, asillustrated in Scheme I. However, as will be recognized by skilledartisans, the identity of the R³ substituent may alter this reactivity.For example, when R³ is trifluoromethyl, a 50:50 mixture of4N-substituted-4-pyrimidineamine A-4 and the corresponding2N-substituted-2-pyrimidineamine is typically obtained. Theregioselectivity of the reaction can also be controlled by adjusting thesolvent and other synthetic conditions (such as temperature), as iswell-known in the art.

The reactions depicted in Scheme I may proceed more quickly when thereaction mixtures are heated via microwave. When heating in thisfashion, the following conditions can be used: heat to 175° C. inethanol for 5-20 min. in a Smith Reactor (Personal Chemistry, Uppsala,Sweden) in a sealed tube (at 20 bar pressure).

The uracil A-1 starting materials can be purchased from commercialsources or prepared using standard techniques of organic chemistry.Commercially available uracils that can be used as starting materials inScheme I include, by way of example and not limitation, uracil (Aldrich#13,078-8; CAS Registry 66-22-8); 5 bromouracil (Aldrich #85,247-3; CASRegistry 51-20-7; 5 fluorouracil (Aldrich #85,847-1; CAS Registry51-21-8); 5 iodouracil (Aldrich #85,785-8; CAS Registry 696-07-1); 5nitrouracil (Aldrich #85,276-7; CAS Registry 611-08-5); 5(trifluoromethyl)-uracil (Aldrich #22,327-1; CAS Registry 54-20-6).Additional 5-substituted uracils are available from GeneralIntermediates of Canada, Inc., Edmonton, Calif. and/or Interchim, Cedex,France, or can be prepared using standard techniques. A myriad oftextbook references teaching suitable synthetic methods for5-substituted uracils are disclosed herein.

Amines A-3 and A-5 can be purchased from commercial sources or,alternatively, can be synthesized utilizing standard techniques. Forexample, suitable amines can be synthesized from nitro precursors usingstandard chemistry. See also Vogel, 1989, Practical Organic Chemistry,Addison Wesley Longman, Ltd. and John Wiley & Sons, Inc.

Skilled artisans will recognize that in some instances, amines A-3 andA-5 and/or substituent R³ on uracil A-1 include functional groups thatrequire protection during synthesis. The exact identity of anyprotecting group(s) used will depend upon the identity of the functionalgroup being protected, and will be apparent to those of skill in theart. Guidance for selecting appropriate protecting groups, as well assynthetic strategies for their attachment and removal, can be found, forexample, in Greene & Wuts, Protective Groups in Organic Synthesis, 3dEdition, John Wiley & Sons, Inc., New York (1999) and the referencescited therein (hereinafter “Greene & Wuts”).

A specific embodiment of Scheme I utilizing 5-fluorouracil (Aldrich#32,937-1) as a starting material is illustrated in Scheme Ia, below.

In Scheme Ia, X, Y, p, R¹¹, R¹², R¹³, R¹⁴, R², and R⁵ are the same asdefined with respect to Scheme I. Asymmetric2N,4N-disubstituted-5-fluoro-2,4-pyrimidinediamine A-10 can be obtainedby reacting 2,4-dichloro-5-fluoropyrimidine A-8 with one equivalent ofamine A-3 (to yield 2-chloro-N4-substituted-5-fluoro-4-pyrimidineamineA-9) followed by one or more equivalents of amine A-5.

In a certain embodiment, the 2,4-pyrimidinediamine compounds describedherein can be synthesized from substituted or unsubstituted cytosines asillustrated in Schemes IIa and IIb, below.

In Scheme IIa, X, Y, p, R¹¹, R¹², R¹³, R¹⁴, R³, and R⁵ are the same asdefined with respect to Scheme I, and PG represents a protecting group.Referring to Scheme IIa, the C4 exocyclic amine of cytosine A-11 isfirst protected with a suitable protecting group PG to yieldN4-protected cytosine A-12. For specific guidance regarding protectinggroups useful in this context, see Vorbrüggen and Ruh-Pohlenz, 2001,Handbook of Nucleoside Synthesis, John Wiley & Sons, NY, pp. 1-631(“Vorbrüggen”). Protected cytosine A-12 is halogenated at the C2position using a standard dehydrating halogenation reagent understandard conditions to yield 2-chloro-4N-protected-4-pyrimidineamineA-13. Reaction with amine A-5 gives A-14, which on deprotection of theC4 exocyclic amine, gives A-15. Reaction of A-15 with amine A-3 yields2,4-pyrimidinediamine derivative A-6.

In Scheme IIb, X, Y, p, R¹¹, R¹², R¹³, R¹⁴, R³, and R⁵ are the same aspreviously defined with respect to Scheme I and PG represents aprotecting group. Referring to Scheme IIb, cytosine A-11 can be reactedwith amine A-3 or protected amine A-18 to yield N4-substituted cytosineA-16 or A-19, respectively. These substituted cytosines can then behalogenated as previously described, deprotected (in the case ofN4-substituted cytosine A-19) and reacted with amine A-5 to yield a2,4-pyrimidinediamine A-6.

Commercially available cytosines that can be used as starting materialsin Schemes IIa and IIb include, but are not limited to, cytosine(Aldrich #14,201-8; CAS Registry 71-30-7); N4-acetylcytosine (Aldrich#37,791-0; CAS Registry 14631-20-0); 5 fluorocytosine (Aldrich#27,159-4; CAS Registry 2022-85-7); and 5-(trifluoromethyl)-cytosine.Other suitable cytosines useful as starting materials in Schemes IIa areavailable from General Intermediates of Canada, Inc., Edmonton, Calif.and/or Interchim, Cedex, France, or can be prepared using standardtechniques. A myriad of textbook references teaching suitable syntheticmethods are disclosed herein.

In a certain embodiment, the 2,4-pyrimidinediamine compounds describedherein can be synthesized from substituted or unsubstituted2-amino-4-pyrimidinols as illustrated in Scheme III, below.

In Scheme III, X, Y, p, R¹¹, R¹², R¹³, R¹⁴, R³, and R⁵ are the same aspreviously defined with respect to Scheme I and LG is a leaving group.Specific examples of suitable LGs include, but are not limited to,halides, such as chloride and bromide, and sulfonate esters, such asmesylates, triflates, tosylates and the like. Referring to Scheme III,2-amino-4-pyrimidinol A-21 is reacted with arylating agent A-22 to yieldN2-substituted-4-pyrimidinol A-23, which is then halogenated aspreviously described to yield N2-substituted-4-halo-2-pyrimidineamineA-24. Further reaction with amine A-3 affords a 2,4-pyrimidinediaminederivative A-6.

Suitable commercially available 2-amino-4-pyrimidinols A-21 that can beused as starting materials in Scheme III are available from GeneralIntermediates of Canada, Inc., Edmonton, Calif. and/or Interchim, Cedex,France, or can be prepared using standard techniques. A myriad oftextbook references teaching suitable synthetic methods are disclosedherein.

References teaching methods useful for synthesizing pyrimidinesgenerally, as well as starting materials described in Schemes I-III, areknown in the art. For specific guidance, the reader is referred toBrown, D. J., “The Pyrimidines”, in The Chemistry of HeterocyclicCompounds, Volume 16 (Weissberger, A., Ed.), 1962, IntersciencePublishers, (A Division of John Wiley & Sons), New York (“Brown I″);Brown, D. J., “The Pyrimidines”, in The Chemistry of HeterocyclicCompounds, Volume 16, Supplement I (Weissberger, A. and Taylor, E. C.,Ed.), 1970, Wiley-Interscience, (A Division of John Wiley & Sons), NewYork (Brown II”); Brown, D. J., “The Pyrimidines”, in The Chemistry ofHeterocyclic Compounds, Volume 16, Supplement II (Weissberger, A. andTaylor, E. C., Ed.), 1985, An Interscience Publication (John Wiley &Sons), New York (“Brown III”); Brown, D. J., “The Pyrimidines” in TheChemistry of Heterocyclic Compounds, Volume 52 (Weissberger, A. andTaylor, E. C., Ed.), 1994, John Wiley & Sons, Inc., New York, pp. 1-1509(Brown IV”); Kenner, G. W. and Todd, A., in Heterocyclic Compounds,Volume 6, (Elderfield, R. C., Ed.), 1957, John Wiley, New York, Chapter7 (pyrimidines); Paquette, L. A., Principles of Modern HeterocyclicChemistry, 1968, W. A. Benjamin, Inc., New York, pp. 1-401 (uracilsynthesis pp. 313, 315; pyrimidinediamine synthesis pp. 313-316; aminopyrimidinediamine synthesis pp. 315); Joule, J. A., Mills, K. and Smith,G. F., Heterocyclic Chemistry, 3rd Edition, 1995, Chapman and Hall,London, UK, pp. 1-516; Vorbrüggen, H. and Ruh-Pohlenz, C., Handbook ofNucleoside Synthesis, John Wiley & Sons, New York, 2001, pp. 1-631(protection of pyrimidines by acylation pp. 90-91; silylation ofpyrimidines pp. 91-93); Joule, J. A., Mills, K. and Smith, G. F.,Heterocyclic Chemistry, 4th Edition, 2000, Blackwell Science, Ltd,Oxford, UK, pp. 1-589; and Comprehensive Organic Synthesis, Volumes 1-9(Trost, B. M. and Fleming, I., Ed.), 1991, Pergamon Press, Oxford, UK.

It has been discovered that 2,4-pyrimidinediamine compounds describedherein modulate the activity of certain cytokines and can be used totreat an inflammatory disease in a subject. Certain compounds disclosedherein elevate the production of anti-inflammatory cytokines and/ordecrease the production of pro-inflammatory cytokines. Such compoundsare useful in treating a variety of inflammatory conditions, examples ofwhich are discussed below. In one aspect, certain compounds elevate theproduction of IL-10, an anti-inflammatory cytokine. Other disclosedcompounds inhibit IL-23 production. The pro-inflammatory effect of IL-23has been well documented and anti-IL-23 antibody therapy currently isbeing used in the treatment of various inflammatory and autoimmunedisorders. Certain embodiments of the disclosed compounds both boostIL-10 production and inhibit IL-23 production. In one embodiment thedisclosed compounds boost IL-10 production at a concentration where theyinhibit IL-23 production. In certain embodiments the disclosed compoundsinhibit IL-23 production in response to an inflammatory stimulus. Forexample, such IL-23 inhibitory compounds may inhibit IL-23 productionwith an inhibitory concentration (IC₅₀) value of less than about 0.01μM, or even less than about 1 nM, to about 20 μM, such as from about 0.1μM to about 10 μM or from about 0.05 μM to about 1 μM. Disclosedcompounds that boost IL-10 production typically raise IL-10 levels fromless than 2-fold—such as by about 10% to about 30% or from about 20% toabout 90%—to about 10-fold. Certain examples of these compounds do notsignificantly inhibit IL-23, but exert their anti-inflammatory effectprimarily by increasing IL-10 production. In certain embodiments,compounds that increase IL-10 production also increase IL-23 production.Such compounds may also be anti-inflammatory if the compounds increaseIL-10 production more effectively than they increase IL-23 production.Compounds disclosed herein that raise IL-10 levels typically raise IL-10levels with an EC₅₀ of from about 0.01 μM, or even less than about 1 nM,to about 20 μM, such as from about 0.1 μM to about 10 μM or from about0.05 μM to about 1 μM.

In certain embodiments, compounds that inhibit IL-23 production alsoboost IL-10 production. However, certain disclosed compounds may inhibitboth IL-23 production and IL-10 production. Typically such compoundsalso are considered to be anti-inflammatory. For example, in certainembodiments, disclosed anti-inflammatory compounds inhibit IL-23production more effectively than they inhibit IL-10 production, forexample from about 2-fold to about 10-fold more effectively, such asfrom about 2-fold to about 5-fold more effectively. In otherembodiments, the anti-inflammatory compounds inhibit IL-23 production aseffectively as they inhibit IL-10, yet still exert an overallanti-inflammatory effect.

Standard physiological, pharmacological and biochemical procedures areavailable for testing the compounds provided herein to identify thosethat possess anti-inflammatory activity. In vitro and in vivo assaysthat may be used to evaluate the anti-inflammatory activity are known tothose of skill in the art. Procedures suitable for testing the IL-23inhibitory and IL-10 boosting activity of the compounds are alsoavailable, and examples of such procedures are described herein.

The term “inflammatory disorder” or “inflammatory disease” is used torefer to abnormalities associated with inflammation, and comprises alarge group of disorders. An inflammatory disorder can be associatedwith acute inflammation and/or chronic inflammation.

Generally, inflammatory disorders include, but are not limited to,respiratory disorders (including asthma, COPD, chronic bronchitis andcystic fibrosis); cardiovascular related disorders (includingatherosclerosis, post-angioplasty, restenosis, coronary artery diseasesand angina); inflammatory diseases of the joints (including rheumatoidand osteoarthritis); skin disorders (including dermatitis, eczematousdermatitis and psoriasis); post transplantation late and chronic solidorgan rejection; multiple sclerosis; autoimmune conditions (includingsystemic lupus erythematosus, dermatomyositis, polymyositis, Sjogren'ssyndrome, polymyalgia rheumatica, temporal arteritis, Behcet's disease,Guillain Barre, Wegener's granulomatosus, polyarteritis nodosa);inflammatory neuropathies (including inflammatory polyneuropathies);vasculitis (including Churg-Strauss syndrome, Takayasu's arteritis);inflammatory disorders of adipose tissue; autoimmune conditions;proliferative disorders (including Kaposi's sarcoma and otherproliferative disorders of smooth muscle cells); inflammatory boweldiseases (including ulcerative colitis and Crohn's disease); allergicreactions; inflammatory myopathies (including as dermatomyositis,polymyositis, and inclusion body myositis); and leukocyte defects(including Chediak-Higashi syndrome and chronic granulomatous disease).

Respiratory Disorders

Respiratory disorders that may be treated include a disease or disorderof the respiratory system that can affect any part of the respiratorytract. Certain diseases cause respiratory symptoms although the diseasesare initially caused by an infection, such as a cold virus, bronchitis,pneumonia and tuberculosis. Other disorders are caused by irritation ofthe lung tissue, such as, for example, by an allergen. These disordersinclude hay fever and other respiratory allergies and asthma. In certainembodiments, the host is at risk of or suffering from a disorder of thelower airway. These include bronchitis, simple and mucopurulent chronicbronchitis, unspecified chronic bronchitis (including chronic bronchitisNOS, chronic tracheitis and chronic tracheobronchitis), emphysema, otherchronic obstructive pulmonary disease, asthma, status asthmaticus andbronchiectasis.

In asthma, the bronchi and bronchioles are typically temporarilyconstricted and inflamed. Other disorders typically involving lungirritants include emphysema, which can result from multiple factorsincluding: smog, cigarette smoke, infection, and a geneticpredisposition to the condition, laryngitis, lung cancer, respiratorydistress syndrome (RDS), which refers to a group of symptoms thatindicate severe malfunctioning of the lungs affecting adults and infantsand specifically Adult respiratory distress syndrome (ARDS). Chronicrespiratory insufficiency (or chronic obstructive pulmonary disease;COPD) is a prolonged or persistent condition characterized by breathingor respiratory dysfunction resulting in reduced rates of oxygenation orthe ability to eliminate carbon dioxide.

The term “asthma” as used herein includes any asthmatic condition markedby recurrent attacks of paroxysmal dyspnea (i.e., “reversibleobstructive airway passage disease”) with wheezing due to spasmodiccontraction of the bronchi (so called “bronchospasm”). Asthmaticconditions which may be treated or even prevented in accordance withthis invention include allergic asthma and bronchial allergycharacterized by manifestations in sensitized persons provoked by avariety of factors including exercise, especially vigorous exercise(“exercise-induced bronchospasm”), irritant particles (pollen, dust,cotton, cat dander) as well as mild to moderate asthma, chronic asthma,severe chronic asthma, severe and unstable asthma, nocturnal asthma, andpsychologic stresses.

Other respiratory disorders include allergic and non-allergic rhinitisas well as non-malignant proliferative and/or inflammatory disease ofthe airway passages and lungs. Allergic rhinitis means generally anyallergic reaction of the nasal mucosa and includes hay fever (seasonalallergic rhinitis) and perennial rhinitis (non-seasonal allergicrhinitis) which are characterized by seasonal or perennial sneezing,rhinorrhea, nasal congestion, pruritis and eye itching, redness andtearing. Non-allergic rhinitis means eosinophilic nonallergic rhinitiswhich is found in patients with negative skin tests and those who havenumerous eosinophils in their nasal secretions.

Non-malignant proliferative and/or inflammatory diseases of the airwaypassages or lungs means one or more of (1) alveolitis, such as extrinsicallergic alveolitis, and drug toxicity such as caused by, e.g.,cytotoxic and/or alkylating agents; (2) vasculitis such as Wegener'sgranulomatosis, allergic granulomatosis, pulmonary hemangiomatosis andidiopathic pulmonary fibrosis, chronic eosinophilic pneumonia,eosinophilic granuloma and sarcoidoses.

In one embodiment, the use of the compounds of the invention reducessymptoms of these disorders, including cough, shortness of breath, chestpain, wheezing, cyanosis, finger clubbing, stridor (a crowing sound whenbreathing), hemoptysis (coughing up of blood), and respiratory failure.The use of these compounds may reduce respiratory acidosis, due to afailure by the lungs to remove carbon dioxide.

In another embodiment, the use of the compounds improves lung function.

Cardiovascular Related Disorders

In one embodiment, the compounds of the invention are administered to apatient suffering from a cardiovascular disorder related toinflammation. These include, but are not limited to, atherosclerosis,post-angioplasty restenosis, coronary artery diseases and angina.

Generally, cardiovascular disorders are a class of diseases that involvethe heart and/or blood vessels (arteries and veins). While the termtechnically refers to any disease that affects the cardiovascularsystem, it is usually used to refer to those related to atherosclerosis(arterial disease).

Cardiovascular inflammatory disorders include atherosclerosis,post-angioplasty, restenosis, coronary artery diseases, angina, andother cardiovascular diseases. In certain embodiments the disorder is anon-cardiovascular inflammatory disorder such as rheumatoid andosteoarthritis, dermatitis, psoriasis, cystic fibrosis, posttransplantation late and chronic solid organ rejection, eczematousdermatitis, Kaposi's sarcoma, or multiple sclerosis. In yet anotherembodiment, the compounds disclosed herein can be selected to treatanti-inflammatory conditions that are mediated by mononuclearleucocytes. In an alternative embodiment, the compounds can beadministered to treat small vessel disease that is not treatable bysurgery or angioplasty, or other vessel disease in which surgery is notan option. The compounds can also be used to stabilize patients prior torevascularization therapy.

Generally, unstable atherosclerotic plaque is a result of multiplefactors but is commonly characterized by an infiltrate of inflammatorycells. Medical research strongly supports a role for inflammation in thepathogenesis, progression, and disruption of atherosclerotic plaque.Clinical studies have demonstrated systemic markers of inflammation tobe strong predictors of clinical events, and specific treatments ofatherosclerosis and its risk factors have been associated withreductions in inflammatory markers. The majority of cardiovascularevents occur at sites of “nonsignificant” stenosis, as inflammation canlead to instability and rupture of these smaller atheroscleroticplaques, which are more numerous than the “significant,” flow-limitingplaques. In fact, direct visualization of inflammatory cells withinplaques is a predictor of unstable coronary disease. The source ofinflammation is uncertain; various infectious agents have been proposedas a stimulator of this inflammatory process. Smooth muscle cellproliferation is also implicated both in chronic cardiovascularpathologies such as atherosclerosis, and more directly in, for example,post-angioplasty restenosis.

Diseases of arteries, arterioles and capillaries generally includeatherosclerosis, peripheral vascular diseases including Raynaud'ssyndrome, thromboangiitis obliterans (Buerger) and other specifiedperipheral vascular diseases such as intermittent claudication.

Proliferative Disorders

Chronic inflammation is a risk factor for many proliferative disorders.For example, in a variety of diseases, airway smooth muscle massincreases due to the coordinated increase in size (hypertrophy) andnumber (hyperplasia) of airway smooth muscle cells. Myocyte migrationmay also serve to regulate airway smooth muscle mass. For example,chronic cellular inflammation and airway wall remodelling withsubepithelial fibrosis and airway smooth muscle (ASM) cell hyperplasiaare features of chronic asthma. In addition, vascular smooth muscle, andimmune cells are stimulated in cardiovascular disorders.

In particular, inflammation is a risk factor in development of cancers,including colon cancer, and data from experimental and observationalstudies suggest that inflammation acts early in the carcinogenic pathwayof colorectal cancer, possibly promoting the progression of colorectaladenomas to adenocarcinoma.

Other Inflammatory Disorders

In another embodiment, the compounds of the invention may beadministered for the treatment or prophylaxis of an inflammatorydisorder or the joints or connective tissue. These disorders includerheumatoid arthritis, lupus erythematosus, Sjogren's syndrome,scleroderma (systemic sclerosis), dermatomyositis, polychondritis,polymyositis, polymyalgia rheumatica, osteoarthritis, septic arthritis,fibromyalgia, gout, pseudogout, spondyloarthropathies, such asankylosing spondylitis, reactive arthritis (Reiter's syndrome),psoriatic arthropathy, enteropathic spondylitis and reactivearthropathy, vasculitis, such as polyarteritis nodosa, Henoch-Schonleinpurpura, serum sickness, Wegener's granulomatosis, giant cell arteritis,temporal arteritis, Takayasu's arteritis, Behcet's syndrome, Kawasaki'sdisease (mucocutaneous lymph node syndrome) and Buerger's disease(thromboangiitis obliterans). In addition, autoimmune conditions such asacute disseminated encephalomyelitis, Addison's disease, ankylosingspondylitisis, antiphospholipid antibody syndrome, autoimmune hepatitis,Coeliac disease, Crohn's disease, diabetes mellitus, Graves' disease,Guillain-Barre syndrome, Hashimoto's disease, idiopathicthrombocytopenic purpura, Kawasaki's Disease, lupus erythematosus,multiple sclerosis, Myasthenia gravis, opsoclonus myoclonus syndrome,optic neuritis, Ord's thyroiditis, pemphigus, pernicious anaemia,primary biliary cirrhosis, Reiter's syndrome, Sjogren's syndrome,Takayasu's arteritis, temporal arteritis, warm autoimmune hemolyticanemia and Wegener's granulomatosis.

In other embodiments, certain inflammatory skin disorders are treated,such as dermatitis, eczematous dermatitis and psoriasis. In generalinflammatory skin disease is a broad category that includes manyconditions, ranging in severity from mild itching to serious medicalhealth complications. Other conditions that are inflammatory skindisorders include eczema generally, acne and rosacea.

Other disorders may also be treated by administration of compounds ofthe invention. In certain embodiments, the disorder to be treated isselected from post transplantation late and chronic solid organrejection; multiple sclerosis; autoimmune conditions (including systemiclupus erythematosus, dermatomyositis, polymyositis, inflammatoryneuropathies (Guillain Barre, inflammatory polyneuropathies), vasculitis(Wegener's granulomatosus, polyarteritis nodosa), and rare disorderssuch as polymyalgia rheumatica, temporal arteritis, Sjogren's syndrome,Behcet's disease, Churg-Strauss syndrome, and Takayasu's arteritis).

Diabetes

In another embodiment, the compounds of the invention may beadministered for the treatment prophylaxis or delay of onset ofdiabetes, pre-diabetes and related disorders. Related disorders ofdiabetes include, but are not limited to, hyperglycemia, abnormalglucose homeostasis, insulin resistance, Syndrome X, metabolicdisorders, diabetic dyslipidemia. In one embodiment, the disease to betreated or prevented is type 2 diabetes. In one embodiment, patients atrisk for developing diabetes are prophylactically treated to preventonset. Patients with diabetes or at risk for developing diabetes can beidentified through several risk factors. One of the key risk factors isage and obesity. Generally patients who are 45 years or older andoverweight (with a body mass index of 25 or greater) is at risk ofdeveloping diabetes.

Inflammatory arthritis comprises a condition where arthritis is presentbecause of localized joint inflammation. Rheumatoid arthritis, generallyconsidered a type of inflammatory arthritis, involves many joints all ofwhich are damaged to some degree by inflammation and its sequelae.Osteoarthritis, also known as “degenerative arthritis” or “degenerativejoint disease,” is a clinical syndrome in which low-grade inflammationresults in pain in the joints, caused by abnormal wearing of thecartilage that covers and acts as a cushion inside joints anddestruction or decrease of synovial fluid that lubricates those joints.In certain embodiments, the inflammatory disorder described herein is aninflammatory arthritis, including but not limited to rheumatoidarthritis or osteoarthritis. Psoriasis comprises a non-contagiousdisorder which affects the skin and joints. The scaly skin patchescaused by psoriasis, called psoriatic plaques, are areas of inflammationand excessive skin production. In certain embodiments, the inflammatorydisorder described herein is psoriasis.

The terms “treating,” “treatment,” and the like are used herein togenerally mean obtaining a desired pharmacological and physiologicaleffect, and refer to complete elimination as well as to any clinicallyor quantitatively measurable reduction in the inflammatory condition forwhich the subject is being treated. “Treatment” is an interventionperformed for preventing the development or altering the pathology orsymptoms of a disorder. Accordingly, “treatment” refers to boththerapeutic treatment and prophylactic or preventative measures.“Treatment” may also be specified as palliative care.

More specifically, 2,4-pyridinediamine compounds described herein whichare used to treat a subject with an inflammatory disorder are providedin a therapeutically effective amount to prevent the disorder (i.e.,inhibit the onset or occurrence of the disorder and/or cause theclinical symptoms of the disorder not to develop in a mammal that may beexposed to or predisposed to the disorder but does not yet experience ordisplay symptoms of the disorder); inhibit the disorder (i.e., arrest orreduce the development of the disorder or its clinical symptoms); orrelieve the disorder (i.e., cause regression of the disorder or itsclinical symptoms). Subjects in need of treatment include those alreadywith one or more inflammatory disorder as well as those in which one ormore inflammatory disorder is to be prevented.

A “subject in need thereof” refers to any subject or individual whocould benefit from the method of treatment described herein. In certainembodiments, a subject in need thereof is a subject predisposed for thedevelopment of one or more inflammatory disorders; a subject having oneor more inflammatory disorders but not exhibiting any clinical symptoms;or a subject having one or more inflammatory disorders and exhibitingsymptoms of the one or more inflammatory disorders. The “subject in needthereof” refers to a vertebrate, such as a mammal. Mammals include, butare not limited to, humans, other primates, rodents (i.e., mice, rats,and hamsters), farm animals, sport animals and pets. In one embodiment,the subject is a mammal such as a human. In certain embodiments, themethods find use in experimental animals, in veterinary application,and/or in the development of animal models for disease.

As used herein, the term “administering” or “introducing” a compound toa subject means providing the compound to a subject. Methods ofadministering to subjects include any of a number of convenient meansincluding, but not limited to, systemic administration (e.g.,intravenous injection, intraparenteral injection, inhalation,transdermal delivery, oral delivery, nasal delivery, rectal delivery,etc.) and/or local administration (e.g., direct injection into a targettissue, delivery into a tissue via cannula, delivery into a targettissue by implantation of a time-release material, or delivery throughthe skin via a topical composition such as a cream, lotion, or thelike), delivery into a tissue by a pump, etc., intraosseously, in thecerebrospinal fluid, or the like. “Orally delivery” refers toadministration in an oral form, such as in a pharmaceutically acceptablecarrier and/or diluent. Oral delivery includes ingestion of the compoundas well as oral gavage of the drug.

Further modes of administration include buccal, sublingual, vaginal,subcutaneous, intramuscular, intradermal, and aerosol administration.

Modes of administration can include delivery via a sustained releaseand/or controlled release drug delivery formulation and/or device.“Sustained release” refers to release of a drug or an active metabolitethereof into the systemic circulation over a prolonged period of timerelative to that achieved by oral administration of a conventionalformulation of the drug. “Controlled release” is a zero order release;that is, the drug releases over time irrespective of concentration.Single, multiple, continuous or intermittent administration can beeffected.

In one embodiment, a composition comprising one or more2,4-pyrimidinediamine compounds described herein is administered orallyto a subject having an inflammatory arthritis such as osteoarthritis. Inanother embodiment, a composition comprising one or more2,4-pyrimidinediamine compounds described herein is injected directlyinto an affected joint of a subject having an inflammatory arthritissuch as osteoarthritis. In yet another embodiment, a compositioncomprising one or more 2,4-pyrimidinediamine compounds described hereinis administered via a topical formulation applied to the skin proximalto an affected joint of a subject having an inflammatory arthritis suchas osteoarthritis arthritis.

A “therapeutically effective amount” or “pharmaceutically effectiveamount” means the amount of 2,4-pyrimidinediamine compound describedherein that, when administered to a subject for treating an inflammatorydisorder, is sufficient to effect such treatment for the disorder. Thus,a “therapeutically effective amount” is an amount indicated fortreatment while not exceeding an amount which may cause significantadverse effects (at a reasonable benefit/risk ratio) within the scope ofsound medical judgment. The “therapeutically effective amount” will varydepending on the compound, and will also be determined by physical andphysiological factors such the disorder and its severity, and the age,body weight, and/or clinical history of the subject to be treated.Methods for evaluating the effectiveness of therapeutic treatments areknown to those of skill in the art.

Doses to be administered are variable according to the treatment period,frequency of administration, the host, and the nature and severity ofthe disorder. The dose can be determined by one of skill in the artwithout an undue amount of experimentation. The 2,4-pyrimidinediaminecompounds described herein are administered in dosage concentrationssufficient to ensure the release of a sufficient dosage unit into thepatient to provide the desired treatment of the inflammatory disorder.The active ingredients may be administered to achieve therapeutic orprophylactic blood concentrations, such as in vivo plasma concentrationsof the 2,4-pyrimidinediamine compounds described herein of from about0.01 to about 10,000 ng/cc, such as from about 0.01 to about 1,000ng/cc. “Therapeutic or prophylactic blood concentrations” refers tosystemic exposure to a sufficient concentration of a compound or anactive metabolite thereof over a sufficient period of time to effectdisease therapy or to prevent the onset or reduce the severity of adisease in the treated animal.

For example, the methods described herein may use compositions toprovide from about 0.01 to about 100 mg/kg body weight/day of the2,4-pyrimidinediamine compounds described herein, from about 0.01 toabout 10 mg/kg body weight/day of the compounds, or about 30 mg/kg bodyweight/day of the compounds. It will be understood, however, that dosagelevels that deviate from the ranges provided may also be suitable in thetreatment of a given disorder.

The 2,4-pyrimidinediamine compounds described herein may be in any formsuitable for administration. Such administrable forms include tablets,buffered tablets, pills, capsules, enteric-coated capsules, dragees,cachets, powders, granules, aerosols, liposomes, suppositories, creams,lotions, ointments, skin patches, parenterals, lozenges, oral liquidssuch as suspensions, solutions and emulsions (oil-in-water orwater-in-oil), ophthalmic liquids and injectable liquids, or sustained-and/or controlled release forms thereof. The desired dose may beprovided in several increments at regular intervals throughout the day,by continuous infusion, or by sustained and/or controlled releaseformulations, or may be presented as a bolus, electuary or paste.

“Practical dosage regimen” refers to a schedule of drug administrationthat is practical for a patient to comply with. For human patients, apractical dosage regimen for an orally administered drug is likely to bean aggregate dose of less than 10 g/day.

In one embodiment, a pharmaceutical composition or formulationcomprising one or more 2,4-pyrimidinediamine compounds described hereinis prepared by admixture with one or more pharmaceutically acceptablecarriers. Other products may be added, if desired, to maximize compoundpreservation, or to optimize a particular method of delivery. Inaddition, the present methods include use of combination compositionscomprising the 2,4-pyrimidinediamine compounds described herein asdescribed herein in conjunction (combination or alternation) with otheragents suitable for the treatment of inflammatory disorders. In certainembodiments, the combination or alternation can be synergistic.

“Pharmaceutically acceptable carrier” or “diluent” means a carrier thatis useful in preparing a pharmaceutical composition that is generallysafe, neither biologically nor otherwise undesirable, not toxic orotherwise unacceptable commensurate with a reasonable benefit/riskratio, compatible with other ingredients of the formulation, andincludes a carrier that is acceptable for veterinary use as well ashuman pharmaceutical use. “A pharmaceutically acceptable carrier” asused in the specification and claims includes both one and more than onesuch carrier.

A “pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and anti-fungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration of a composition comprising one or more2,4-pyrimidinediamine compounds described herein. Examples of suchcarriers or diluents include, but are not limited to, water, saline,Ringer's solutions and dextrose solution. The volume of thepharmaceutical composition is based on the intended mode ofadministration and the safe volume for the individual patient, asdetermined by a medical professional.

The present invention relates to use of the 2,4-pyrimidinediaminecompounds described herein for the manufacture of a medicament.

The foregoing disclosure is further described in the followingnon-limiting examples. In the examples below as well as throughout theapplication, the following abbreviations have the following meanings. Ifnot defined, the terms have their generally accepted meanings.

mL=milliliter

s=singlet

d=doublet

t=triplet

q=quartet

m=multiplet

dd=doublet of doublets

br=broad

nM=nanomolar

μg=microgram

ng=nanogram

MS=mass spectrum or mass spectrometry

LC=liquid chromatography

DMSO=dimethylsulfoxide

μL=microliter

mM=millimolar

rpm=revolutions per minute

EXAMPLES Example 1 Synthesis of5-Fluoro-N2-[4-(4-methylpiperazino)-3-trifluoromethyl]phenyl-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)-2,4-pyrimidinediamine

2-Fluoro-5-nitrobenzotrifluoride (2 g) and 1-methylpiperazine (2 mL)were dissolved in methanol (5 mL). The yellow solution was stirred atroom temperature overnight. The reaction mixture was diluted with water(100 mL) and extracted with ethyl acetate (2×100 mL). The organicsolutions were evaporated to give2-(4-methylpiperazino)-5-nitrobenzotrifluoride.

2-(4-Methylpiperazino)-5-nitrobenzotrifluoride was dissolved in methanol(100 mL) and to the solution was added 10% Pd—C. The reaction mixturewas reacted under hydrogen atmosphere (˜40 psi) for 1 h. The catalystwas filtered off over celite and washed with methanol. The filtrate wasevaporated to give [4-(4-methylpiperazino)-3-trifluoromethyl]aniline(2.25 g, 91% in two steps). ¹H NMR (DMSO-d6): δ 2.19 (s, 3H), 2.38 (br,4H), 2.70 (t, J=4.5 Hz, 4H), 5.31 (br, 2H), 6.73 (dd, J=2.4, 8.7 Hz,1H), 6.78 (d, J=2.7 Hz, 1H), 7.20 (d, J=8.1 Hz, 1H).

4-Amino-1,2,2,6,6-pentamethylpiperidine (1 g) and2,6-dichloro-5-fluoropyrimidine (1.5 g) were dissolved in methanol (10mL). The reaction solution was stirred at room temperature overnight.The reaction solution was evaporated and crystallized from ethyl acetateand hexanes to give2-chloro-5-fluoro-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)-4-pyrimidineamineHCl salt (1.65 g, 93%). ¹H NMR (DMSO-d6): δ 1.38 (s, 6H), 1.48 (s, 6H),2.02 (m, 4H), 2.68 (d, J=4.8 Hz, 3H), 4.33 (br, 1H), 8.10 (d, J=3.3 Hz,1H), 8.32 (d, J=6.9 Hz, 1H), 9.66 (br, 1H).

2-Chloro-5-fluoro-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)-4-pyrimidineamine(300 mg) and [4-(4-methylpiperazino)-3-trifluoromethyl]aniline (300 mg)were suspended in isopropanol (1 mL) and TFA (5 drops). The solution washeated at 100° C. overnight, then cooled to room temperature. Thesolution was evaporated and purified by flash column chromatography (2.0M NH₃/MeOH in dichloromethane=2, 4, 6, 10%) to give5-fluoro-N2-[4-(4-methylpiperazino)-3-trifluoromethyl]phenyl-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)-2,4-pyrimidinediamine(440 mg, 84%). ¹H NMR (DMSO-d6): δ 1.04 (s, 6H), 1.07 (s, 6H), 1.44 (t,J=11.7 Hz, 2H), 1.68 (d, J=9.9 Hz, 2H), 2.18 (s, 3H), 2.20 (s, 3H), 2.41(br, 4H), 2.76 (t, J=4.2 Hz, 4H), 4.29 (br, 1H), 7.16 (d, J=8.4 Hz, 1H),7.32 (d, J=8.4 Hz, 1H), 7.75 (d, J=2.1 Hz, 1H), 7.84 (d, J=3.6 Hz, 1H),8.07 (d, J=8.7 Hz, 1H), 9.13 (s, 1H); 19F NMR (282 MHz, DMSO-d6):δ−165.87, −59.89; LCMS: purity: 100%; MS (m/e): 524.43 (MH+).

The following compounds were made in a similar fashion or by methodsdescribed herein or known to those of skill in the art of organicsynthesis:

Example 25-Fluoro-N2-[4-(4-methylsulfonylpiperazino)-3-trifluoromethyl]phenyl-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)-2,4-pyrimidinediamine

LCMS: purity: 100%; MS (m/e): 588.49 (MH+); ¹H NMR (DMSO-d₆): δ 1.04 (s,6H), 1.07 (s, 6H), 1.44 (t, J=12.0 Hz, 2H), 1.68 (d, J=10.2 Hz, 2H),2.18 (s, 3H), 2.86 (t, 4H), 2.93 (s, 3H), 3.19 (t, 4H), 4.32 (br, 1H),7.19 (d, J=7.2 Hz, 1H), 7.40 (d, J=8.1 Hz, 1H), 7.80 (s, 1H), 7.85 (d,1H), 8.08 (d, J=9.3 Hz, 1H), 9.18 (s, 1H); ¹⁹F NMR (282 MHz, DMSO-d₆):δ−59.87, −165.68.

Example 35-Fluoro-N2-[4-(4-methylsulfonyl)piperazin-1-yl)-3-(trifluoromethyl)]phenyl-N4-(1,2,2,5,5-pentamethylpyrrolidin-3-yl)-2,4-pyrimidinediamine

LCMS: purity: 97.82%; MS (m/e): 574.34 (MH+); ¹H NMR (DMSO-d6): δ 0.93(s, 3H), 1.10-1.12 (m, 9H), 1.75-1.85 (m, 2H), 2.15 (s, 3H), 2.55 (d,J=12.0 Hz, 4H), 2.90 (s, 3H), 3.75 (d, J=11.8 Hz, 4H), 4.44 (br, 1H),6.60 (m, 2H), 6.75 (m, 1H), 7.32 (s, 1H), 8.01 (d, J=4.3 Hz, 1H), 9.12(s, 1H).

Example 45-Fluoro-N2-[4-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)]phenyl-N4-(1,2,2,5,5-pentamethylpyrrolidin-3-yl)-2,4-pyrimidinediamine

LCMS: purity: 95.20%; MS (m/e): 510.59 (MH+); ¹H NMR (DMSO-d6): δ 0.92(s, 3H), 1.10-1.15 (m, 9H), 1.75-1.85 (m, 2H), 2.10 (s, 3H), 2.25 (s,3H), 2.50 (d, J=10.8 Hz, 4H), 3.73 (d, J=11.2 Hz, 4H), 4.54 (m, 1H),6.56 (m, 2H), 6.72 (m, 1H), 7.28 (s, 1H), 8.11 (d, J=4.5 Hz, 1H), 9.27(s, 1H).

Example 5N2-[3-Chloro-4-(4-methylsulfonylpiperazino)]phenyl-5-fluoro-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)-2,4-pyrimidinediamine

LCMS: purity: 99.56%; MS (m/e): 554.20 (MH+); ¹H NMR (DMSO-d₆): δ1.08(s, 12H), 1.46 (t, J=12.0 Hz, 2H), 1.68 (d, J=10.5 Hz, 2H), 2.18 (s,3H), 2.93 (s, 3H), 2.96 (t, 4H), 3.25 (t, 4H), 4.33 (br, 1H), 7.01 (d,J=8.7 Hz, 1H), 7.18 (d, J=8.4 Hz, 1H), 7.60 (d, J=9.3 Hz, 1H), 7.79 (s,1H), 7.83 (d, J=3.9 Hz, 1H), 9.04 (s, 1H); ¹⁹F NMR (282 MHz, DMSO-d₆):δ−166.22.

Example 6N2-[3-Chloro-4-(4-methylpiperazino)]phenyl-5-fluoro-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)-2,4-pyrimidinediamine

LCMS: purity: 97.41%; MS (m/e): 490.10 (MH+); ¹H NMR (DMSO-d₆): δ 1.07(s, 12H), 1.45 (t, J=12.0 Hz, 2H), 1.67 (d, J=11.4 Hz, 2H), 2.18 (s,3H), 2.21 (s, 3H), 2.45 (br, 4H), 2.85 (br, 4H), 4.32 (br, 1H), 6.95 (d,J=9.0 Hz, 1H), 7.16 (d, J=8.1 Hz, 1H), 7.57 (d, J=8.4 Hz, 1H), 7.72 (d,J=1.8 Hz, 1H), 7.82 (d, J=3.6 Hz, 1H), 8.98 (s, 1H); ¹⁹F NMR (282 MHz,DMSO-d₆): δ−166.42.

Example 7N2-[3-Chloro-4-(4-methylpiperazino)sulfonyl]phenyl-5-fluoro-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)-2,4-pyrimidinediamine

LCMS: purity: 98.98%; MS (m/e): 554.13 (MH+); ¹H NMR (DMSO-d₆): δ1.40(s, 6H), 1.44 (s, 6H), 1.82 (t, 2H), 2.10 (d, J=14.1 Hz, 2H), 2.74 (br,4H), 3.09 (br, 4H), 4.48 (br, 1H), 7.75 (m, 3H), 7.96 (s, 1H), 8.02 (d,J=3.3 Hz, 1H), 8.59 (br, 1H), 9.76 (s, 1H); ¹⁹F NMR (282 MHz, DMSO-d₆):δ−163.36.

Example 8N2-[3-Chloro-4-piperazinosulfonyl]phenyl-5-fluoro-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)-2,4-pyrimidinediamine

LCMS: purity: 99.38%; MS (m/e): 540.03 (MH+); ¹H NMR (DMSO-d₆): δ1.34(br, 12H), 3.04 (br, 4H), 3.65 (br, 4H), 4.28 (br, 1H), 6.31 (s, 1H),6.53 (d, J=10.5 Hz, 1H), 6.66 (s, 1H), 7.54 (d, J=8.7 Hz, 1H), 7.78 (s,1H).

Example 95-Fluoro-N2-[4-(4-methylsulfonylpiperazino)-3-trifluoromethyl]phenyl-N4-(2,2,6,6-tetramethylpiperidin-4-yl)-2,4-pyrimidinediamine

LCMS: purity: 98.98%; MS (m/e): 574.50 (MH+); ¹H NMR (DMSO-d₆): δ1.33(s, 6H), 1.40 (s, 6H), 1.52 (t, J=12.6 Hz, 2H), 1.94 (d, J=10.2 Hz, 2H),2.87 (t, 4H), 2.93 (s, 3H), 3.20 (br, 4H), 4.42 (br, 1H), 7.46 (d, J=8.1Hz, 1H), 7.51 (d, J=8.1 Hz, 1H), 7.80 (s, 1H), 7.92 (d, J=3.6 Hz, 1H),8.02 (d, J=8.7 Hz, 1H), 9.24 (s, 1H); ¹⁹F NMR (282 MHz, DMSO-d₆):δ−165.53, −59.87.

Example 10N2-[4-(4-Cyclopropylsulfonylpiperazino)-3-trifluoromethyl]phenyl-5-fluoro-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)-2,4-pyrimidinediamine

LCMS: purity: 96.79%; MS (m/e): 614.26 (MH+); ¹H NMR (DMSO-d₆): δ 0.94(m, 2H), 1.02 (d, J=8.4 Hz, 2H), 1.19 (s, 12H), 1.58 (t, J=12.3 Hz, 2H),1.84 (d, J=12.6 Hz, 2H), 2.41 (s, 3H), 2.68 (m, 1H), 2.86 (t, 4H), 4.36(br, 1H), 7.38 (d, 1H), 7.41 (d, J=8.1 Hz, 1H), 7.79 (s, 1H), 7.88 (d,J=3.6 Hz, 1H), 8.07 (d, J=8.7 Hz, 1H), 9.22 (s, 1H); ¹⁹F NMR (282 MHz,DMSO-d₆): δ−59.87, −165.61.

Example 11N2-[3-Cyano-4-(4-methylsulfonylpiperazino)]phenyl-5-fluoro-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)-2,4-pyrimidinediamine

LCMS: purity: 97.55%; MS (m/e): 545.27 (MH+); ¹H NMR (DMSO-d₆): δ 1.28(s, 12H), 1.66 (t, J=12.0 Hz, 2H), 1.91 (d, 2H), 2.54 (s, 3H), 2.95 (s,3H), 3.09 (t, 4H), 4.38 (br, 1H), 7.10 (d, J=8.7 Hz, 1H), 7.45 (d, 1H),7.81 (d, J=9.0 Hz, 1H), 7.90 (d, J=3.6 Hz, 1H), 7.97 (s, 1H), 9.17 (s,1H); ¹⁹F NMR (282 MHz, DMSO-d₆): δ−165.75.

Example 12N2-[3-Difluoromethoxy-4-(4-methylsulfonylpiperazino)]phenyl-5-fluoro-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)-2,4-pyrimidinediamine

MS (m/e) 586.14 (MH⁺); ¹H NMR (DMSO-d₆): δ 9.03 (s, 1H), 7.87-7.86 (d,J=2.4 Hz, 1H), 7.56-7.53 (d, J=9.0 Hz, 1H), 7.46 (s, 1H), 7.39 (bs, 1H),7.28-6.78 (t, J=76.5 Hz 1H), 6.98-6.95 (d, J=8.4 Hz, 1H), 4.43 (bm, 1H),3.23 (s, 4H), 2.95 (s, 4H), 2.92 (s, 3H), 1.88 (bs, 2H), 1.64 (bs,J=11.7 Hz, 2H), 1.26 (s, 12H).

Example 13N2-(3-Difluoromethoxy-4-methoxy)phenyl-5-fluoro-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)-2,4-pyrimidinediamine

MS (m/e) 454.11 (MH⁺); ¹H NMR (DMSO-d₆): δ 8.91 (s, 1H), 8.15 (s, 1H),7.82-7.81 (d, J=3.9 Hz, 1H), 7.57-7.54 (d, J=8.7 Hz, 1H), 7.49 (s, 1H),7.18-6.69 (t, J=74.7 Hz 1H), 7.16 (s, 1H), 6.95-6.92 (d, J=9.0 Hz, 1H),4.38 (bm, 1H), 3.73 (s, 3H), 2.25 (s, 3H), 1.73-1.69 (d, J=12.0 Hz, 2H),1.53-1.45 (t, J=12.3 Hz, 2H), 1.11 (s, 12H).

Example 14N2-[3-Chloro-4-(4-cyclopropylsulfonylpiperazino)]phenyl-5-fluoro-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)-2,4-pyrimidinediamine

LCMS: purity: 96.45%; MS (m/e): 580.13 (MH+); ¹H NMR (DMSO-d₆): δ 0.95(m, 2H), 1.02 (d, J=7.8 Hz, 2H), 1.14 (s, 12H), 1.52 (t, J=12.0 Hz, 2H),1.76 (d, J=10.8 Hz, 2H), 2.30 (s, 3H), 2.66 (m, 1H), 2.95 (t, 4H), 3.32(t, 4H), 4.35 (br, 1H), 7.01 (d, J=9.0 Hz, 1H), 7.24 (d, J=6.9 Hz, 1H),7.58 (dd, J=8.7 Hz, 1H), 7.77 (s, 1H), 7.84 (d, J=3.9 Hz, 1H), 9.03 (s,1H); ¹⁹F NMR (282 MHz, DMSO-d₆): δ−166.19

Example 15N2-(2,2-Difluoro-4-ethyl-benzoxazin-3-one-7-yl)-5-fluoro-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)-2,4-pyrimidinediamine

MS (m/e) 493.20 (MH+); ¹H NMR (DMSO-d₆): δ 9.33 (s, 1H), 8.01 (s, 1H),7.88 (s, 1H), 7.37-7.25 (m, 3H), 4.41 (s, 1H), 3.99 (bs, 2H), 2.25 (s,3H), 1.74-1.70 (d, J=12.3 Hz, 2H), 1.57-1.49 (t, J=12.3 Hz, 2H), 1.14(bs, 15H).

Example 165-Ethoxycarbonyl-N2-[4-(4-methylsulfonylpiperazino)-3-trifluoromethyl]phenyl-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)-2,4-pyrimidinediamine

LCMS: purity: 98.99%; MS (m/e): 642.23 (MH+); ¹H NMR (DMSO-d₆): δ 1.11(s, 6H), 1.14 (s, 6H), 1.29 (t, J=6.9 Hz, 3H), 1.38 (t, J=12.6 Hz, 2H),1.91 (d, J=10.8 Hz, 2H), 2.31 (s, 3H), 2.88 (t, 4H), 2.94 (s, 3H), 3.21(t, 4H), 4.23 (q, J=6.9 Hz, 2H), 4.34 (br, 1H), 7.46 (d, J=9.0 Hz, 1H),7.76 (br, 1H), 8.08 (d, J=7.2 Hz, 1H), 8.16 (d, J=7.8 Hz, 1H), 8.57 (s,1H), 9.96 (br, 1H); ¹⁹F NMR (282 MHz, DMSO-d₆): δ−59.94.

Example 17N2-[3-Chloro-4-(4-methylsulfonylpiperazino)]phenyl-5-ethoxycarbonyl-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)-2,4-pyrimidinediamine

LCMS: purity: 97.85%; MS (m/e): 608.12 (MH+); ¹H NMR (DMSO-d₆): δ 1.13(s, 12H), 1.29 (t, J=6.9 Hz, 3H), 1.36 (t, J=11.1 Hz, 2H), 1.88 (d,J=12.3 Hz, 2H), 2.28 (s, 3H), 2.93 (s, 3H), 2.98 (t, 4H), 3.26 (t, 4H),4.22 (q, J=6.9 Hz, 2H), 4.41 (br, 1H), 7.05 (d, J=8.4 Hz, 1H), 7.73 (m,2H), 8.09 (d, 1H), 8.55 (s, 1H), 9.82 (br, 1H).

Example 18N2-[4-(4-Methylsulfonylpiperazino)-3-trifluoromethyl]phenyl-5-nitro-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)-2,4-pyrimidinediamine

LCMS: purity: 90.54%; MS (m/e): 615.19 (MH+); ¹H NMR (DMSO-d₆): δ1.02(s, 6H), 1.11 (s, 6H), 1.54 (t, J=11.4 Hz, 2H), 1.81 (d, J=10.2 Hz, 2H),2.25 (s, 3H), 2.89 (br, 4H), 2.93 (s, 3H), 3.21 (br, 4H), 4.44 (br, 1H),7.50 (d, J=9.0 Hz, 1H), 7.73 (s, 1H), 8.11 (s, 1H), 8.39 (br, 1H), 8.98(s, 1H), 10.49 (br, 1H); ¹⁹F NMR (282 MHz, DMSO-d₆): 6-59.98.

Example 19N2-[3-Chloro-4-(4-methylsulfonylpiperazino)]phenyl-5-nitro-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)-2,4-pyrimidinediamine

LCMS: purity: 94.77%; MS (m/e): 581.10 (MH+); ¹H NMR (DMSO-d₆): δ 1.32(s, 6H), 1.43 (s, 6H), 2.05 (m, 4H), 2.70 (d, J=3.9 Hz, 3H), 2.94 (s,3H), 3.00 (t, 4H), 3.27 (t, 3H), 4.60 (m, 1H), 7.11 (d, J=8.1 Hz, 1H),7.64 (m, 2H), 8.48 (d, J=7.5 Hz, 1H), 8.99 (s, 1H), 9.23 (br, 1H), 10.42(s, 1H)

Example 205-Cyano-N2-[4-(4-methylsulfonylpiperazino)-3-trifluoromethyl]phenyl-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)-2,4-pyrimidinediamine

LCMS: purity: 89.22%; MS (m/e): 595.44 (MH+); ¹H NMR (DMSO-d₆): δ1.00(s, 6H), 1.07 (s, 6H), 1.57 (m, 4H), 2.19 (s, 3H), 2.88 (t, 4H), 2.93(s, 3H), 3.20 (t, 4H), 4.34 (br, 1H), 7.44 (d, J=8.7 Hz, 2H), 7.70 (br,1H), 8.10 (d, 1H), 8.32 (s, 1H), 9.91 (br, 1H); ¹⁹F NMR (282 MHz,DMSO-d₆): δ−59.95

Example 21N2-[3-Chloro-4-(4-methylpiperazino)]phenyl-5-cyano-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)-2,4-pyrimidinediamine

LCMS: purity: 86.57%; MS (m/e): 497.30 (MH+); ¹H NMR (DMSO-d₆): δ 1.01(s, 6H), 1.05 (s, 6H), 1.58 (m, 4H), 2.16 (s, 3H), 2.21 (s, 3H), 2.88(t, 4H), 4.40 (br, 1H), 6.99 (d, J=7.8 Hz, 1H), 7.37 (d, 1H), 7.61 (d,2H), 8.28 (s, 1H), 9.75 (br, 1H).

Example 225-Fluoro-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)-N2-[4-(pyridin-4-yl)-3-trifluoromethyl]phenyl-2,4-pyrimidinediamine

LCMS: purity: 93.72%; MS (m/e): 503.33 (MH+); ¹H NMR (DMSO-d₆): δ1.05(s, 6H), 1.07 (s, 6H), 1.46 (t, J=11.7 Hz, 2H), 1.70 (d, J=11.7 Hz, 2H),2.17 (s, 3H), 4.32 (br, 1H), 7.18 (d, J=8.7 Hz, 1H), 7.29 (d, J=6.0 Hz,3H), 7.90 (d, J=3.9 Hz, 1H), 7.99 (s, 1H), 8.27 (d, J=8.4 Hz, 1H), 8.59(d, J=6.0 Hz, 2H), 9.46 (s, 1H); ¹⁹F NMR (282 MHz, DMSO-d₆): δ−164.93,−56.23.

Example 23N2-[4-(4-Methylsulfonylpiperazino)-3-trifluoromethyl]phenyl-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)-5-trifluoromethyl-2,4-pyrimidinediamine

LCMS: purity: 98.14%; MS (m/e): 638.20 (MH+); ¹H NMR (DMSO-d₆): δ1.34(s, 12H), 1.91 (br, 4H), 2.88 (t, 4H), 2.93 (s, 3H), 3.21 (t, 4H), 4.62(br, 1H), 7.49 (d, 1H), 7.67 (br, 1H), 8.03 (d, 1H), 8.22 (s, 1H), 9.74(br, 1H); ¹⁹F NMR (282 MHz, DMSO-d₆): δ−59.94, −60.77.

Example 24N2-[3-Chloro-4-(4-methylsulfonylpiperazino)]phenyl-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)-5-trifluoromethyl-2,4-pyrimidinediamine

LCMS: purity: 91.02%; MS (m/e): 603.91 (MH+); ¹H NMR (DMSO-d₆): δ1.05(s, 6H), 1.08 (s, 6H), 1.61 (m, 4H), 2.21 (s, 3H), 2.93 (s, 3H), 2.97(t, 4H), 3.30 (t, 4H), 4.54 (br, 1H), 6.40 (d, 1H), 7.03 (d, 1H), 7.64(m, 2H), 8.16 (s, 1H); ¹⁹F NMR (282 MHz, DMSO-d₆): δ−60.76.

Example 25 In Vitro IL-10 and IL-23 Assays

Abbreviations

THP-1: human acute monocyte leukemia cell line

IFNγ: interferon gamma

SAC: Staphylococcus aureus cells, heat-killed and formalin-fixed

IKK2VI inhibitor: (5-phenyl-2-ureido)thiophene-3-carboxamide

A. Screening Protocol

Materials

THP-1 cells and RPMI growth media containing 1% P/S and 10% FBS

IFNγ (Peprotech, Cat No. 300-02)

White clear bottom 96 well plates (Fisher, Cat No. 07-200-587, Corning#3903)

SAC (12% solution from Calbiochem, Cat No. 507858)

CELL TITER GLO® reagent (Promega, Cat No. G7573)

Positive controls, IKK2VI inhibitor (Calbiochem, Cat No. 401-483)

Exponentially growing THP-1 cells were plated (100K/well in 100 μL) instandard RPMI media (1% P/S+10% FBS) containing 50 ng/mL IFNγ (1000U/mL) onto a white clear bottom 96 well plate and leave the cells in 37°C. incubator for 24 h.

After 24 h incubation, 100 μL of RPMI media containing 2× concentratedtest compound per well was added to the above cell-culture media (finalconcentration of test compound was from 0.01 μM to 20 μM). The plateswere pre-incubated for 1 h at 37° C. before being stimulated with SAC.

After 1 h compound pre-incubation, 10 μL per well of 20× concentratedSAC solution in RPMI media was added to give a final concentration of0.01%, the plates were shaken and incubated at 37° C. for an additional18 h.

155 μL of the supernatant was harvested from each well and to theremaining 50 μL/well of the cell culture plate was added 50 μL of CELLTITER GLO® reagent. The plates were incubated for 1-2 minutes on ashaker and read for luminescence intensity to determine the compoundcytotoxicity.

The cell culture supernatant collected above was used to carry out IL23ELISA (65 μL-Supernatant) and IL10 ELISA (90 μL-Supernatant) asdescribed below.

B. Human IL-23 (p19/p40) ELISA Protocol (e-Biosciences)

Materials

96-well high binding opaque white plates (from Pierce, Cat No. 15042)

1×PBS; 1×TBST washing buffer

Blocking Solution: 0.5% Casein in PBS (from BDH, Cat No. 440203H)

Dilution Solution: 1% BSA in PBS (10% BSA from Fisher, Cat No. 37525)

Capture antibody: Rat anti-human IL-23 (p19) (e-Biosciences, Cat. No.14-7238-85)

Detection antibody: Primary Mouse Biotinylated anti-human IL-12(p40/p70) (e-biosciences, Cat No. 13-7129-85); Secondary HRP-conjugatedStreptavidin (R&D Systems, Cat No. DY998).

rHuman-IL-23 (e-biosciences, Cat No. 34-8239)

(Suggested starting concentration=5 ng/mL in RPMI cell culture media)

Cell Culture Supernatant (65 μL from THP-1 cells primed with IFNγ (50ng/mL-1000 U/mL) and stimulated with 0.01% SAC)

SUPERSIGNAL® ELISA Pico Chemiluminescent substrate [Pierce, Cat No.37069]

Coating Plates:

To 10.5 mL PBS was added 50 μL of anti-IL23 (p19) capture antibody (2.5μg/mL), mixed well and 100 μL of the coating solution was added to eachwell of the 96 well white plates from Pierce, cover and incubateovernight at 4° C.

Blocking the Plates:

The anti-IL23 (p19)-antibody-coated plates were washed 2× using TBST(use plate washer) and the plates were blocked using 200 μL of 0.5%Casein for 1.5-2 h at room temperature with shaking.

Addition of Supernatant and Detection:

The plates were washed 2× using TBST and the supernatant (65 μL/well)was transferred to the above pre-blocked/IL23(p19)-antibody-coated 96well plate and incubated at RT for 1.5 h with shaking.

The plate was washed 4× using TBST (plate washer) and 100 μL/well ofdetection antibody solution was added. The detection antibody solutionwas prepared from 2 μL of biotin labeled anti-IL-12 (p40/p70) antibodyin 11 mL 1% BSA/PBS solution (1-5000 dilution). The plates wereincubated for 1 hour with shaking at RT.

Again, the plate was washed 4× with TBST and 100 μL of HRP labeledStreptavidin (R&D Systems) solution (10 μL/10 mL 1% BSA solution) wasadded and incubated at RT for another 45 min with shaking.

After 45 min, the plate was washed with TBST 4× and 100 μL/well SuperSignal ELISA Pico Chemiluminescent Substrate from Pierce (3.5 mL A+3.5mL B+3.5 mL MQ water) was added, the plate was shaken for 1-2 minutesthen read on a plate reader to quantify the amount of IL-23 secreted bythe cells.

C. Human IL-10 ELISA Protocol (e-Biosciences)

Materials

96-well high binding opaque white plates (from Pierce, Cat No. 15042)

1×PBS; 1×TBST washing buffer

Blocking Solution: 0.5% Casein in PBS (from BDH, Cat No. 440203H)

Dilution Solution: 1% BSA in PBS (10% BSA from Fisher, Cat No. 37525)

Capture antibody: Rabbit anti-human IL-10 (e-Biosciences, Cat. No.14-7108-85)

Detection antibody: Primary Mouse Biotinylated anti-human IL-10(e-biosciences, Cat No. 13-7109-85); Secondary HRP-conjugatedStreptavidin (R&D Systems, Cat No. DY998).

rHuman-IL-10 (e-biosciences, Cat No. 34-8109)

(Suggested starting concentration=1 ng/mL in RPMI cell culture media)

Cell Culture Supernatant (90 μL from THP-1 cells primed with IFNγ (50ng/mL-1000 U/mL) and stimulated with 0.01% SAC)

SUPERSIGNAL® ELISA Pico Chemiluminescent substrate [Pierce, Cat No.37069]

Coating Plates:

To 10.5 mL PBS was added 50 μL of anti-IL10 capture antibody (2.5μg/mL), mixed well and 100 μL of the coating solution was added to eachwell of the 96 well white plates from Pierce. The plates were coveredand incubated overnight at 4° C.

Blocking the Plates:

The anti-IL10 antibody-coated plates were washed 2× using TBST (useplate washer) and blocked the using 200 μL of 0.5% Casein for 1.5-2 h atRT with shaking.

Addition of Supernatant and Detection:

The plates were washed 2× using TBST and the supernatant (100 μL/well)was transferred to the above pre-blocked/IL10-antibody-coated 96 wellplate and incubated at RT for 1.5 h with shaking.

The plate was washed 4× using TBST (plate washer) and 100 μL/welldetection antibody solution prepared from 2 μL of biotin labeledanti-IL-10 antibody in 11 mL 1% BSA/PBS solution (1-5000 dilution) wasadded. The plates were incubated for 1 hour with shaking at RT.

Again, the plate was washed 4× with TBST and 100 μL of HRP labeledStreptavidin (R&D Systems) solution (10 μL/10 mL 1% BSA solution) wasadded and incubated at RT for another 45 min with shaking.

After 45 min, the plate was washed with TBST 4× and 100 uL/well SuperSignal ELISA Pico Chemiluminescent Substrate from Pierce (3.5 mL A+3.5mL B+3.5 mL MQ water) was added, the plate was shaken for 1-2 minutesthen read on a plate reader to quantify the amount of IL-10 secreted bythe cells.

D. Results

Data of compounds from this assay are reported in Table 1 below:

TABLE 1 STRUCTURE IL-10 IL-23 1

+ − 2

− − 3

+ − 4

− − 5

+ not tested 6

+ not tested 7

+ − 8

+ − 9

+ not tested 10

+ not tested 11

+ not tested 12

+ not tested 13

+ not tested 14

+ not tested 15

+ not tested 16

+ not tested 17

− not tested 18

+ − 19

+ − 20

+ not tested 21

+ not tested 22

+ not tested 23

+ − 24

+ not tested 25

+ − 26

+ not tested Table 1 shows the effect of the tested compounds onexpression levels of IL-10 and IL-23 in THP-1 cells stimulated with SAC.“+” indicates an increase in production, “−” indicates a decrease inproduction, and “not tested” indicates that the sample was not tested.

Example 26 Methods of Treatment

A. A compound described herein having anti-inflammatory activity isidentified by a suitable method. A subject having an inflammatorydisorder is identified by a suitable method. The subject is treated withthe compound described herein by administering the compound in a carriersuitable for the chosen mode of administration in an amount effectivefor treating the inflammatory disorder.

B. A compound described herein having anti-inflammatory activity isidentified by a suitable method. A subject having psoriasis isidentified by a suitable method. The subject is treated with thecompound described herein by administering the compound in a carriersuitable for the chosen mode of administration in an amount effectivefor treating the psoriasis.

C. A compound described herein having anti-inflammatory activity isidentified by a suitable method. A subject having Crohn's disease isidentified by a suitable method. The subject is treated with thecompound described herein by administering the compound in a carriersuitable for the chosen mode of administration in an amount effectivefor treating the Crohn's disease.

D. A compound described herein having anti-inflammatory activity isidentified by a suitable method. A subject having osteoarthritis isidentified by a suitable method. The subject is treated with thecompound described herein by administering the compound in a carriersuitable for the chosen mode of administration in an amount effectivefor treating the osteoarthritis.

E. A compound described herein that inhibits the production of IL-23 isidentified by a suitable method. A subject having an inflammatorydisorder is identified by a suitable method. The subject is treated withthe compound described herein by administering the compound in a carriersuitable for the chosen mode of administration in an amount effectivefor treating the inflammatory disorder.

F. A compound described herein that increases the production of IL-10 isidentified by a suitable method. A subject having an inflammatorydisorder is identified by a suitable method. The subject is treated withthe compound described herein by administering the compound in a carriersuitable for the chosen mode of administration in an amount effectivefor treating the inflammatory disorder.

G. A compound described herein that inhibits the production of IL-23 andincreases the production of IL-10 is identified by a suitable method. Asubject having an inflammatory disorder is identified by a suitablemethod. The subject is treated with the compound described herein byadministering the compound in a carrier suitable for the chosen mode ofadministration in an amount effective for treating the inflammatorydisorder.

H. A compound described herein that inhibits the production of IL-23 andinhibits the production of IL-10 is identified by a suitable method. Asubject having an inflammatory disorder is identified by a suitablemethod. The subject is treated with the compound described herein byadministering the compound in a carrier suitable for the chosen mode ofadministration in an amount effective for treating the inflammatorydisorder.

I. A compound described herein that increases the production of IL-10and increases the production of IL-23 is identified by a suitablemethod. A subject having an inflammatory disorder is identified by asuitable method. The subject is treated with the compound describedherein by administering the compound in a carrier suitable for thechosen mode of administration in an amount effective for treating theinflammatory disorder.

While the invention has been described with reference to specificembodiments, variations and modifications may be made without departingfrom the spirit and the scope of the invention. Such variations andmodifications are to be considered within the purview and scope of theinvention as defined by the appended claims.

All of the above-mentioned references are herein incorporated byreference in their entirety to the same extent as if each individualreference was specifically and individually indicated to be incorporatedherein by reference in its entirety.

We claim:
 1. A method for inhibiting the production of IL-23, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of one or more compounds according to the formula:

wherein X represents (CH₂)_(m), wherein m is an integer from 1 to 5, andwherein one or more CH₂ are optionally replaced with O, S or N(R⁰),wherein R⁰ represents H, C₁-C₇ alkyl or C₃-C₈ cycloalkyl; Y representsN(R¹), O or S, wherein R¹ represents H, C₁-C₇ alkyl or C₃-C₈ cycloalkyl;R¹¹, R¹², R¹³, and R¹⁴ each independently represents C₁-C₇ alkyl; prepresents 0 or 1; R² represents H, C₁-C₇ alkyl or C₂-C₈ alkanoyl; R³represents H, halogen, cyano, nitro, (C₁-C₇ alkoxy)carbonyl, C₁-C₇alkyl, C₁-C₇ haloalkyl, C₁-C₇ alkoxy or C₁-C₇ haloalkoxy; R⁴ representsH, C₁-C₇ alkyl or C₂-C₈ alkanoyl; and R⁵ represents a substituted arylor heteroaryl.
 2. The method of claim 1, wherein the method furthercomprises stimulating the production of IL-10 and/or wherein the subjectis a human.
 3. A method for stimulating production of IL-10, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of one or more compounds according to the formula:

wherein X represents (CH₂)_(m), wherein m is an integer from 1 to 5, andwherein one or more CH₂ are optionally replaced with O, S or N(R⁰),wherein R⁰ represents H, C₁-C₇ alkyl or C₃-C₈ cycloalkyl; Y representsN(R¹), O or S, wherein R¹ represents H, C₁-C₇ alkyl or C₃-C₈ cycloalkyl;R¹¹, R¹², R¹³, and R¹⁴ each independently represents C₁-C₇ alkyl; prepresents 0 or 1; R² represents H, C₁-C₇ alkyl or C₂-C₈ alkanoyl; R³represents H, halogen, cyano, nitro, (C₁-C₇ alkoxy)carbonyl, C₁-C₇alkyl, C₁-C₇ haloalkyl, C₁-C₇ alkoxy or C₁-C₇ haloalkoxy; R⁴ representsH, C₁-C₇ alkyl or C₂-C₈ alkanoyl; and R⁵ represents a substituted arylor heteroaryl.